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Isolation of dental pulp stem cells with high osteogenic potential

Abstract

Dental pulp stem cells/progenitor cells (DPSCs) can be easily obtained and can have excellent proliferative and mineralization potentials. Therefore, many studies have investigated the isolation and bone formation of DPSCs. In most previous reports, human DPSCs were traditionally isolated by exploiting their ability to adhere to plastic tissue culture dishes. DPSCs isolated by plastic adherence are frequently contaminated by other cells, which limits the ability to investigate their basic biology and regenerative properties. Additionally, the proliferative and osteogenic potentials vary depending on the isolated cells. It is very difficult to obtain cells of a sufficient quality to elicit the required effect upon transplantation. Considering clinical applications, stem cells used for regenerative medicine need to be purified in order to increase the efficiency of bone regeneration, and a stable supply of these cells must be generated. Here, we review the purification of DPSCs and studies of cranio-maxillofacial bone regeneration using these cells. Additionally, we introduce the prospective isolation of DPSCs using specific cell surface markers: low-affinity nerve growth factor and thymocyte antigen 1.

Background

Dental pulp, which contains connective tissue, mesenchymal cells, neural fibers, blood vessels, and lymphatics, is located at the center of the pulp chamber enclosed in mineralized dentin. The main functions of dental pulp are to produce dentin and to maintain the biological and physiological vitality of dentin [1]. Dental pulp stem cells/progenitor cells (DPSCs) in adult dental pulp tissue are induced to differentiate into odontoblasts to form reparative dentin in order to protect dental pulp [2, 3]. DPSCs and stem cells from human exfoliated deciduous teeth (SHEDs) have a high proliferative potential, an extensive self-renewal ability, and a multilineage differentiation capacity, with osteogenic, chondrogenic, adipogenic, neurogenic, and myogenic potentials [3,4,5]. In particular, DPSCs and SHEDs have a high mineralization potential and are considered to be useful in bone regenerative therapy [6,7,8]. Many studies regarding DPSCs have been reported because dental pulp tissue is easily obtained. In most previous reports, DPSCs were traditionally isolated by exploiting their ability to adhere to plastic tissue culture dishes [3]. However, adherent culture conditions on plastic dishes inevitably change the expression of surface markers and the biological properties of stem cells. Consequently, stem cell properties may diminish during adherent culture on plastic tissue culture dishes [9, 10]. Furthermore, DPSCs isolated based on their adherence to plastic are frequently contaminated by cells with different phenotypes. Additionally, the proliferative and osteogenic potentials vary depending on the isolated cells. It is very difficult to obtain cells of a sufficient quality to elicit the required effect upon transplantation. Considering clinical applications, stem cells used for regenerative medicine need to be purified in order to increase the efficiency of bone regeneration, and a stable supply of these cells must be generated. Here, we review the purification of DPSCs and the studies of cranio-maxillofacial bone regeneration using these cells. Additionally, we introduce the prospective isolation of DPSCs with high osteogenic potential.

Bone regenerative therapy in the cranio-maxillofacial region

Bone regenerative therapies are required to treat many diseases affecting the cranio-maxillofacial region such as craniofacial abnormalities, bone defects following mandible tumor surgery, trauma, jaw bone necrosis, and bone augmentation for dental implants. Bone regeneration plays significant roles in the recovery of function and improvement of aesthetic disorders in the cranio-maxillofacial area. Autogenous bones harvested from the patient’s own body, such as the iliac bone, scapula, and fibula, have been used for major reconstruction of the maxillofacial area [11]. This bone grafting requires large-scale surgery, e.g., reconstruction using vascular pedicle bone grafts and particulate cancellous bone marrow with a titanium mesh [11, 12]. Autogenous bone from the chin and ramus of the mandible, allogenic bone, and xenogenic bone have been used for minor bone augmentation [13, 14].

Regenerative medicine studies have used various approaches such as osteoinductive chemical factors, osteoinductive growth factors, osteoinductive materials, extracellular matrix, and cell-based tissue engineering. Many studies of adult stem cell-based tissue engineering have sought to effectively regenerate bone in the maxillofacial area. One recent line of progress in stem cell research is bone regeneration using stem cells from bone marrow (BMMSCs). BMMSCs not only have high osteogenic and chondrogenic potentials, but also have an excellent regenerative potential to treat bone defects in vivo [15]. Therefore, these cells are considered to be very useful for bone regenerative therapies in the maxillofacial area. Several groups showed that tissue-engineered bone constructed with BMMSCs elicits beneficial effects in a mandibular defect model, a maxillary sinus floor elevation model, and a jaw malformation model [16,17,18]. In humans, injectable tissue-engineered bone formation using BMMSCs and platelet-rich plasma was applied to 14 cases for ridge augmentation and dental implant placement [19]. Furthermore, another group applied BMMSCs seeded onto β-tricalciumphosphate to upper jaw bone defects for dental implant placement after trauma [20].

Dental stem cells are an attractive option for regenerative therapy because they can be easily expanded to generate the number required for generation of graft materials. Furthermore, dental stem cells can be easily obtained in comparison with BMMSCs because exfoliated deciduous teeth and impacted third molar teeth are often extracted for clinical or orthodontic reasons. All dental stem cells (including DPSCs, SHEDs, periodontal ligament stem cells, dental follicle stem cells, and stem cells isolated from the apical papilla) are considered to be obtained via minimally invasive methods when isolated from these extracted teeth. They can give rise to proliferative cells and osteogenic cells under appropriate conditions [3,22,, 4, 21–23].

Characterization of stem cells from dental pulp

DPSCs are traditionally isolated from dental pulp by exploiting their ability to adhere to plastic tissue culture dishes after enzyme digestion [3] (Fig. 1a). This technique gives rise to heterogeneous cell populations that are frequently contaminated by other cells, including osteoblasts, osteoprogenitor cells, fat cells, reticular cells, macrophages, endothelial cells, and hematopoietic cells. There is a pressing need to enrich regenerative DPSCs. The study of DPSCs has been profoundly influenced by earlier studies of BMMSCs because DPSCs are positive for cell surface markers similar to those of BMMSCs, including CD44, CD73, CD105, STRO-1, and CD146, but are negative for CD45, CD34, CD14, C11b, CD79, CD19, and HLA-DR [5]. SHEDs also highly express MSC markers, including CD105, CD146, STRO-1, and CD29, but are negative for CD31 and CD34 [5]. Various methods have been tested to isolate and purify clonal subsets of stem cells from dental pulp, including immunoselection of cell surface markers by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) (Table 1).

Fig. 1
figure 1

a Traditional isolation of dental pulp stem/progenitor cells (DPSCs) by adherent culture on dishes. b Prospective isolation of DPSCs by flow cytometric identification of cell surface markers. c Representative fluorescence-activated cell sorting profiles of dental pulp cells. d A representative phase-contrast micrograph of plastic-adherent colony-forming LNGFRLow+THY-1High+ cells with fibroblast morphologies. Scale bars = 100 μm

Table 1 Purification of dental pulp stem/progenitor cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs)

DPSCs were first isolated from dental pulp tissue using cell surface markers, mainly STRO-1. Several studies reported that STRO-1+ cells have a high colony-forming ability and a multilineage differentiation capability [4,25,, 24–26] and express CD146, and a pericyte marker (3G5) in perivascular and perineural sheath regions [24]. STRO-1+ and CD146+ cells in pulp of deciduous teeth are also located in perivascular regions [4]. c-Kit+CD34+CD45− cells isolated from dental pulp by flow cytometry have a potent proliferative potential and readily differentiate into osteogenic precursors capable of generating three-dimensional woven bone tissue chips in vitro [27]. Although STRO-1+c-Kit+CD34+ human DPSCs (hDPSCs), which reside in a perivascular niche, have a lower proliferative capacity than STRO-1+c-Kit+CD34− hDPSCs; they strongly express Nestin and the surface antigen low-affinity nerve growth factor (LNGFR, also called CD271) [28]. STRO-1+c-Kit+CD34+ hDPSCs show a stronger tendency toward neurogenic commitment than STRO-1+c-Kit+CD34− hDPSCs, even though no significant differences between the two subpopulations arise after differentiation toward mesoderm lineages (osteogenic, adipogenic, and myogenic). c-Kit+FLK-1+CD34+STRO-1+ stem cells isolated from a plastic-adherent population by FACS have a potent growth potential (92% colony formation from 3–4 seeded cells) and are multipotent [9]. Other groups have demonstrated that colony-derived populations of DPSCs express typical mesenchymal markers, including CD29, CD44, CD90, CD166, and CD105 [29].

Subsequently, a side population (SP) was isolated from dental pulp based on efflux of the fluorescent dye Hoechst 33342 detected by FACS [30, 31]. This method, which has been used on SP cell populations from hematopoietic bone marrow, highly enriches cells with stem cell activity [32]. SP cells from dental pulp exhibit a self-renewal capacity with a long proliferative lifespan and differentiate into odontoblast-like cells, neurons, chondrocytes, and adipocytes [30, 31]. Furthermore, CD31−CD146− SP cells and CD105+ cells from dental pulp have high proliferative and migration activities and a multilineage differentiation potential in vitro, including adipogenic, dentinogenic, angiogenic, and neurogenic potentials [33, 34]. In a whole dental pulp removal model, transplantation of canine CD31−CD146− SP and CD105+ DPSCs expressing angiogenic and neurotrophic factors promotes regeneration of pulp in permanent teeth [33, 35]. Immature dental pulp stem cells express various embryonic stem cell markers [36]. A recent study of SHEDs demonstrated that stage-specific embryonic antigen-4+ cells derived from human deciduous dental pulp tissue have a multilineage differentiation potential in vitro [37].

Dental pulp originates from migrating neural crest cells; therefore, stem cells have been isolated from dental pulp using LNGFR, an embryonic neural crest marker [38, 39]. LNGFR has been used to prospectively isolate neural crest stem cells (NCSCs) from mammalian fetal peripheral nerves [40]. NCSCs can self-renew and differentiate into neurons, Schwann cells, and smooth muscle-like myofibroblasts in vitro. The characteristics of NCSCs are similar to those of MSCs. Cranial neural crest-derived cells contribute to ectomesenchymal cells in the developing dental papilla during tooth development [41, 42]. Cranial neural crest-derived LNGFR+ ectomesenchymal stem cells have odonto-differentiation potential [43]. Multipotent NCSCs have been identified not only in the early embryonic stage, but also in adulthood. Neural crest-related stem cells were isolated from mature dental pulp in several studies [39, 44, 45]. The enriched cell population expresses Nestin, LNGFR, and SOX10 and can be induced to differentiate into osteoblasts, melanocytes, and Schwann cells [45]. Thymocyte antigen 1 (THY-1, also called CD90)+ glial cells generate multipotent MSCs that produce dental pulp cells and odontoblasts [46]. LNGFR+THY-1+ neural crest-like cells derived from human pluripotent stem cells can differentiate into both mesenchymal and neural crest lineages [47]. Therefore, LNGFR and THY-1 could be useful to isolate clonogenic DPSCs from neural crest-derived dental pulp tissue.

Prospective isolation of DPSCs using surface makers

Although many methods to enrich DPSCs have been devised, most assume that plastic-adherent cells are stem cells. Adherent culture on plastic dishes inevitably changes the expression of surface markers and gradually diminishes the differentiation, proliferation, and migration potencies of stem cells [9, 10]. These methods may not be able to reproduce the experimental results or reveal the biological properties of DPSCs. It is important to establish a method that can be used to prospectively isolate purified DPSC populations without cell culture. Therefore, specific cell surface markers need to be identified in order to isolate highly regenerative DPSCs. LNGFR and THY-1 have been identified as selective markers for the purification and phenotypic characterization of MSCs from various sources such as bone marrow, decidua, adipose tissue, and synovium [48, 49]. Especially in human bone marrow, LNGFR+THY-1+ cells are extremely enriched with clonogenic cells (2 × 105-fold enrichment vs. whole bone marrow cells) [48]. Our study demonstrated that these markers can also be used to prospectively isolate hDPSC populations, thereby avoiding the need for prolonged cell culture [50] (Fig. 1b). Flow cytometric analyses revealed five cell populations, namely, LNGFR+THY-1+, LNGFRLow+THY-1High+, LNGFR−THY-1Low+, LNGFR+THY-1−, and LNGFR−THY-1− (Fig. 1c). Although LNGFR+THY-1+ cells in bone marrow exhibit the highest clonogenic potential [48], assessment of the number of colonies showed that LNGFRLow+THY-1High+ cells in dental pulp have a significantly higher colony-forming potential than LNGFR+THY-1+ cells [50]. LNGFRLow+THY-1High+ cells are uniformly small and have a spindle-shaped (MSC-like) morphology (Fig. 1d). The cell population considered to be DPSCs comprises two cell types, and it seems that purity can be increased by selecting one of these. However, a LNGFRLow+THY-1High+ cell population was not observed in FACS profiles of human BMMSCs stained with anti-LNGFR and anti-THY-1 antibodies [48]. The discrepancy of the expression pattern of cell surface markers between dental pulp tissue and bone marrow tissue may be due to differences in the origin of the cells. Dental pulp tissue is thought to be derived from migrating neural crest cells, whereas bone marrow tissue originates from the mesoderm and neural crest [51, 52]. During development, neural crest cells from the dorsal neural tube migrate to various locations and divide into four main functional domains, namely, the cranial neural crest, the trunk neural crest, the vagal and sacral neural crest, and the cardiac neural crest. Neural crest cells differentiate into a vast range of cells, including neurons and glial cells of the peripheral nervous system, smooth muscle cells, bone, and cartilage cells. Each distinct cell type responds to specific migration and differentiation signals to generate the appropriate cells and tissues [53]. Therefore, the phenotypes and biological properties of each cell type may differ.

Biological properties of stem cells from dental pulp

DPSCs and SHEDs have a high proliferation rate and a multilineage differentiation capability, including osteogenic, chondrogenic, adipogenic, neurogenic, and myogenic potentials [3,4,5]. Osteogenic differentiation of DPSCs is easily induced in vitro by adding dexamethasone, ascorbic acid, and β-glycerophosphate to culture medium supplemented with fetal bovine serum [54, 55]. DPSCs express bone markers such as alkaline phosphatase, type 1 collagen, osteocalcin, and osteonectin under osteogenic induction [3, 56]. DPSCs have a faster population doubling time and a higher mineralization potential than BMMSCs [6, 7]. SHEDs have a higher proliferation rate and a higher capability for osteogenic differentiation than BMMSCs and even DPSCs [4, 57]. Overall, DPSCs and SHEDs are more suitable than BMMSCs for mineralized tissue regeneration. In our study, prospectively isolated LNGFRLow+THY-1High+ DPSCs showed a high clonogenic potential and a multipotent differentiation capability for mesenchymal lineages (Fig. 2a). The adipogenic, osteogenic, and chondrogenic capacities of LNGFRLow+THY-1High+ cells were higher than those of LNGFR+THY-1+ cells (Fig. 2a, b) [50]. Interestingly, the proliferation rates of LNGFRLow+THY-1High+ cells and LNGFR+THY-1+ cells did not significantly differ at early passages. Therefore, cultured hDPSCs isolated from crude dental pulp cells contain two cell types that originate from LNGFRLow+THY-1High+ and LNGFR+THY-1+ cells. High LNGFR expression may inhibit differentiation of hDPSCs into osteoblasts and adipocytes [38], while low LNGFR expression might maintain the stemness of hDPSCs in the dental pulp microenvironment. THY-1+ dental pulp cells localized in the sub-odontoblastic layer can differentiate into hard tissue-forming cells and may thus provide a source of odontoblastic cells [58]. THY-1+ human adipose-derived stromal cells show osteogenic potential in vitro and significantly increase bone formation in a calvarial defect model [59]. THY-1+ cells in other tissues also show a high proliferative capacity and osteogenic potential [60, 61]. These reports suggest that THY-1 is important to isolate stem cell-like cells with a potent mineralization potential. LNGFRLow+THY-1High+ DPSCs display a high proliferation rate and a long-term survival using a transillumination procedure such as cranial windows when transplanted into cranial defects of immunodeficient mice [50]. Therefore, LNGFRLow+THY-1High + cells can increase the cell viability in cell transplantation, and this is considered to be advantage for differentiation into osteoblasts and secretion of each growth factor to promote bone morphogenesis. For successful tissue engineering, formation of blood vessels toward the transplanted tissue is required for transportation of oxygen and nutrients to the transplanted cells. When transplanted, stem cells such as DPSCs promote angiogenesis for bone regeneration in the maxillofacial region. DPSCs have a paracrine effect by stimulating the formation of blood vessels in the host tissue through secretion of angiogenic factors [62,63,64,65,66,67,68]. Furthermore, DPSCs and SHEDs may have stronger immunomodulatory properties and high anti-apoptotic activity [69,70,71,72,73,74,75,76]. Thus, DPSCs and SHEDs could also have potential for clinical applications in autologous stem cell transplantation for bone regenerative therapy.

Fig. 2
figure 2

a Adipogenic (Adipo), osteogenic (Osteo), and chondorogenic (Chondro) differentiation of LNGFRLow+THY-1High+ cells. Scale bars = 100 μm. b Adipogenic (Adipo), osteogenic (Osteo), and chondorogenic (Chondro) differentiation of LNGFR+THY-1+ cells. Scale bars = 100 μm

Studies of bone regeneration in the cranio-maxillofacial region using stem cells from dental pulp

There are many studies of bone regeneration using DPSCs and SHEDs in the cranio-maxillofacial region in vivo because these cells have high osteogenic potential (Table 2). Several studies reported that transplantation of expanded DPSCs and SHEDs with scaffolds, such as fibroin, collagen membrane, and hydroxyapatite/tricalcium phosphate ceramic particles, repairs critical-size cranial bone defects of mice and rats [8, 77, 78]. Yamada et al. demonstrated that cell-based therapy using stem cells derived from deciduous teeth and dental pulp of puppies together with platelet-rich plasma can induce new bone formation in critical-size mandibular bone defects [79]. Ito et al. demonstrated that the high osteogenic ability of DPSCs contributes to the osseointegration of dental implants [80]. Alkaisi et al. reported that SHEDs can enhance bone consolidation in a rabbit mandibular distraction model [81]. A study of a large animal model showed that stem cells from deciduous teeth of miniature pigs regenerate bone to repair critical-size swine mandible bone defects [82]. In terms of clinical applications of DPSCs in humans, a biocomplex constructed from DPSCs and a collagen sponge scaffold was reported to be useful for bone tissue repair in human mandibular bone defects after extraction of third molars [83]. However, these cells might have been contaminated by non-regenerative cells with a poor bone-formation ability because these studies did not use purified cells.

Table 2 Studies of bone regeneration by stem cells from dental pulp in the cranio-maxillofacial region in vivo

Several studies investigated bone formation using hDPSCs purified by MACS for the repair of bone defects. Pisciotta et al. reported that STRO-1+ hDPSCs cultured in human serum-containing medium repair critical-size parietal bone defects in immunocompromised rats [84]. Giuliani et al. reported that CD34+ hDPSCs together with a collagen sponge regenerate compact bone with uniform vascularization after tooth extraction [85]. Ricco et al. reported that CD34+c-kit+STRO-1+ hDPSCs with fibroin scaffolds induce mature bone formation and repair critical-size bone defects in immunocompromised rats [86].

In our study, LNGFRLow+THY-1High+ and LNGFR+THY-1+ cells prospectively isolated by FACS were transplanted into critical-sized calvarial defects to evaluate their therapeutic potential [50]. LNGFRLow+THY-1High+ hDPSCs exhibit long-term survival and osteoblastic differentiation in immunohistochemical analyses. Microcomputed tomography-guided morphometric analysis showed that LNGFRLow+THY-1High+ cells induce the highest level of bone regeneration after transplantation into calvarial defects. The bone-formation potential of LNGFRLow+THY-1High+ cells is markedly higher than that of LNGFR+THY-1+ cells. Therefore, traditionally cultured DPSCs isolated from crude dental pulp cells are considered to comprise two cell types, namely, highly osteogenic cells and lowly osteogenic cells. We believe that enrichment of regenerative cells will lead to successful bone regenerative therapy through high levels of engraftment, survival, and proliferation post-transplantation.

Conclusions

Considering clinical applications for bone regeneration, cell-based therapy using DPSCs requires a prolonged period of culture to obtain a sufficient number of cells for transplantation because only a small number of DPSCs can be obtained from a single tooth. Therefore, it is important to stabilize the quality and quantity of transplanted cells by ensuring they have high proliferative and osteogenic capabilities. Cultured DPSCs isolated from crude dental pulp cells are considered to comprise two cell types: regenerative and non-regenerative cells. Hence, isolation of the optimal cell population for bone regeneration is important for regenerative therapy. There is a pressing need to identify selective markers of DPSCs with high osteogenic potential. LNGFR and THY-1 can be used to prospectively isolate a pure population of DPSCs from human dental pulp by FACS. However, purification of DPSCs using these markers is still insufficient compared with that of BMMSCs. Consequently, it is necessary to further enhance their purity by using additional markers. Furthermore, specific markers of other easily obtained dental stem cells should be identified to acquire a cell source for cranio-maxillofacial bone regeneration in a future study because DPSCs cannot be obtained from non-vital teeth.

Abbreviations

BMMSCs:

Stem cells from bone marrow

CM:

Conditioned media

DPSCs:

Dental pulp stem cells/progenitor cells

FACS:

Fluorescence-activated cell sorting

hDPSCs:

Human dental pulp stem cells/progenitor cells

LNGFR:

Low-affinity nerve growth factor

MACS:

Magnetic-activated cell sorting

MSCs:

Mesenchymal stem cells

NCSCs:

Neural crest stem cells

SHEDs:

Stem cells from human exfoliated deciduous teeth

SP:

Side population

THY-1:

Thymocyte antigen 1

References

  1. Liu H, Gronthos S, Shi S. Dental pulp stem cells. Methods Enzymol. 2006;419:99–113.

    Article  CAS  PubMed  Google Scholar 

  2. Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos S, Robey PG, Shi S. Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J Dent Res. 2003;82:976–81.

    Article  CAS  PubMed  Google Scholar 

  3. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97:13625–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100:5807–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009;88:792–806.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Alge DL, Zhou D, Adams LL, Wyss BK, Shadday MD, Woods EJ, et al. Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model. J Tissue Eng Regen Med. 2010;4:73–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Jensen J, Tvedesoe C, Rolfing JH, Foldager CB, Lysdahl H, Kraft DC, et al. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model. SICOT J. 2016;2:16.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Seo BM, Sonoyama W, Yamaza T, Coppe C, Kikuiri T, Akiyama K, et al. SHED repair critical-size calvarial defects in mice. Oral Dis. 2008;14:428–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. d’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, et al. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ. 2007;14:1162–71.

    Article  PubMed  Google Scholar 

  10. Yu J, He H, Tang C, Zhang G, Li Y, Wang R, et al. Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging. BMC Cell Biol. 2010;11:32.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Cannon TY, Strub GM, Yawn RJ, Day TA. Oromandibular reconstruction. Clin Anat. 2012;25:108–19.

    Article  PubMed  Google Scholar 

  12. Yamada H, Nakaoka K, Sonoyama T, Kumagai K, Ikawa T, Shigeta Y, et al. Clinical usefulness of mandibular reconstruction using custom-made titanium mesh tray and autogenous particulate cancellous bone and marrow harvested from tibia and/or ilia. J Craniofac Surg. 2016;27:586–92.

    Article  PubMed  Google Scholar 

  13. Nkenke E, Neukam FW. Autogenous bone harvesting and grafting in advanced jaw resorption: morbidity, resorption and implant survival. Eur J Oral Implantol. 2014;7(Suppl 2):S203–217.

    PubMed  Google Scholar 

  14. Wu J, Li B, Lin X. Histological outcomes of sinus augmentation for dental implants with calcium phosphate or deproteinized bovine bone: a systematic review and meta-analysis. Int J Oral Maxillofac Surg. 2016;45:1471–7.

    Article  CAS  PubMed  Google Scholar 

  15. Petite H, Viateau V, Bensaid W, Meunier A, de Pollak C, Bourguignon M, et al. Tissue-engineered bone regeneration. Nat Biotechnol. 2000;18:959–63.

    Article  CAS  PubMed  Google Scholar 

  16. Zhao J, Hu J, Wang S, Sun X, Xia L, Zhang X, et al. Combination of beta-TCP and BMP-2 gene-modified bMSCs to heal critical size mandibular defects in rats. Oral Dis. 2010;16:46–54.

    Article  CAS  PubMed  Google Scholar 

  17. Xia L, Xu Y, Chang Q, Sun X, Zeng D, Zhang W, et al. Maxillary sinus floor elevation using BMP-2 and Nell-1 gene-modified bone marrow stromal cells and TCP in rabbits. Calcif Tissue Int. 2011;89:53–64.

    Article  CAS  PubMed  Google Scholar 

  18. Zhang D, Chu F, Yang Y, Xia L, Zeng D, Uludag H, et al. Orthodontic tooth movement in alveolar cleft repaired with a tissue engineering bone: an experimental study in dogs. Tissue Eng Part A. 2011;17:1313–25.

    Article  CAS  PubMed  Google Scholar 

  19. Ueda M, Yamada Y, Kagami H, Hibi H. Injectable bone applied for ridge augmentation and dental implant placement: human progress study. Implant Dent. 2008;17:82–90.

    Article  PubMed  Google Scholar 

  20. Rajan A, Eubanks E, Edwards S, Aronovich S, Travan S, Rudek I, et al. Optimized cell survival and seeding efficiency for craniofacial tissue engineering using clinical stem cell therapy. Stem Cells Transl Med. 2014;3:1495–503.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod. 2008;34:166–71.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Kato T, Hattori K, Deguchi T, Katsube Y, Matsumoto T, Ohgushi H, et al. Osteogenic potential of rat stromal cells derived from periodontal ligament. J Tissue Eng Regen Med. 2011;5:798–805.

    Article  CAS  PubMed  Google Scholar 

  23. Honda MJ, Imaizumi M, Suzuki H, Ohshima S, Tsuchiya S, Satomura K. Stem cells isolated from human dental follicles have osteogenic potential. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;111:700–8.

    Article  PubMed  Google Scholar 

  24. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res. 2003;18:696–704.

    Article  PubMed  Google Scholar 

  25. Yang X, van den Dolder J, Walboomers XF, Zhang W, Bian Z, Fan M, et al. The odontogenic potential of STRO-1 sorted rat dental pulp stem cells in vitro. J Tissue Eng Regen Med. 2007;1:66–73.

    Article  CAS  PubMed  Google Scholar 

  26. Yang X, Zhang W, van den Dolder J, Walboomers XF, Bian Z, Fan M, et al. Multilineage potential of STRO-1+ rat dental pulp cells in vitro. J Tissue Eng Regen Med. 2007;1:128–35.

    Article  CAS  PubMed  Google Scholar 

  27. Laino G, Carinci F, Graziano A, d’Aquino R, Lanza V, De Rosa A, et al. In vitro bone production using stem cells derived from human dental pulp. J Craniofac Surg. 2006;17:511–5.

    Article  PubMed  Google Scholar 

  28. Pisciotta A, Carnevale G, Meloni S, Riccio M, De Biasi S, Gibellini L, et al. Human Dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations. BMC Dev Biol. 2015;15:14.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Gandia C, Arminan A, Garcia-Verdugo JM, Lledo E, Ruiz A, Minana MD, et al. Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction. Stem Cells. 2008;26:638–45.

    Article  PubMed  Google Scholar 

  30. Honda MJ, Nakashima F, Satomura K, Shinohara Y, Tsuchiya S, Watanabe N, et al. Side population cells expressing ABCG2 in human adult dental pulp tissue. Int Endod J. 2007;40:949–58.

    Article  CAS  PubMed  Google Scholar 

  31. Iohara K, Zheng L, Ito M, Tomokiyo A, Matsushita K, Nakashima M. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. Stem Cells. 2006;24:2493–503.

    Article  CAS  PubMed  Google Scholar 

  32. Matsuzaki Y, Kinjo K, Mulligan RC, Okano H. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity. 2004;20:87–93.

    Article  CAS  PubMed  Google Scholar 

  33. Iohara K, Imabayashi K, Ishizaka R, Watanabe A, Nabekura J, Ito M, et al. Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1. Tissue Eng Part A. 2011;17:1911–20.

    Article  CAS  PubMed  Google Scholar 

  34. Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, et al. A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp. Stem Cells. 2008;26:2408–18.

    Article  PubMed  Google Scholar 

  35. Iohara K, Zheng L, Ito M, Ishizaka R, Nakamura H, Into T, et al. Regeneration of dental pulp after pulpotomy by transplantation of CD31(−)/CD146(−) side population cells from a canine tooth. Regen Med. 2009;4:377–85.

    Article  CAS  PubMed  Google Scholar 

  36. Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes Massironi SM, Pereira LV, et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs. 2006;184:105–16.

    Article  CAS  PubMed  Google Scholar 

  37. Kawanabe N, Fukushima H, Ishihara Y, Yanagita T, Kurosaka H, Yamashiro T. Isolation and characterization of SSEA-4-positive subpopulation of human deciduous dental pulp cells. Clin Oral Investig. 2015;19:363–71.

    Article  PubMed  Google Scholar 

  38. Mikami Y, Ishii Y, Watanabe N, Shirakawa T, Suzuki S, Irie S, et al. CD271/p75(NTR) inhibits the differentiation of mesenchymal stem cells into osteogenic, adipogenic, chondrogenic, and myogenic lineages. Stem Cells Dev. 2011;20:901–13.

    Article  CAS  PubMed  Google Scholar 

  39. Waddington RJ, Youde SJ, Lee CP, Sloan AJ. Isolation of distinct progenitor stem cell populations from dental pulp. Cells Tissues Organs. 2009;189:268–74.

    Article  PubMed  Google Scholar 

  40. Morrison SJ, White PM, Zock C, Anderson DJ. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 1999;96:737–49.

    Article  CAS  PubMed  Google Scholar 

  41. Chai Y, Jiang X, Ito Y, Bringas Jr P, Han J, Rowitch DH, et al. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000;127:1671–9.

    CAS  PubMed  Google Scholar 

  42. Deng MJ, Jin Y, Shi JN, Lu HB, Liu Y, He DW, et al. Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch. Tissue Eng. 2004;10:1597–606.

    Article  CAS  PubMed  Google Scholar 

  43. Xing Y, Nie X, Chen G, Wen X, Li G, Zhou X, et al. Comparison of P75 NTR-positive and -negative etcomesenchymal stem cell odontogenic differentiation through epithelial-mesenchymal interaction. Cell Prolif. 2016;49:185–94.

    Article  CAS  PubMed  Google Scholar 

  44. Pan W, Kremer KL, Kaidonis X, Ludlow VE, Rogers ML, Xie J, et al. Characterization of p75 neurotrophin receptor expression in human dental pulp stem cells. Int J Dev Neurosci. 2016;53:90–8.

    Article  CAS  PubMed  Google Scholar 

  45. Al-Zer H, Apel C, Heiland M, Friedrich RE, Jung O, Kroeger N, et al. Enrichment and schwann cell differentiation of neural crest-derived dental pulp stem cells. In Vivo. 2015;29:319–26.

    CAS  PubMed  Google Scholar 

  46. Kaukua N, Shahidi MK, Konstantinidou C, Dyachuk V, Kaucka M, Furlan A, et al. Glial origin of mesenchymal stem cells in a tooth model system. Nature. 2014;513:551–4.

    Article  CAS  PubMed  Google Scholar 

  47. Ouchi T, Morikawa S, Shibata S, Fukuda K, Okuno H, Fujimura T, et al. LNGFR + THY-1+ human pluripotent stem cell-derived neural crest-like cells have the potential to develop into mesenchymal stem cells. Differentiation. 2016;92:270–80.

    Article  CAS  PubMed  Google Scholar 

  48. Mabuchi Y, Morikawa S, Harada S, Niibe K, Suzuki S, Renault-Mihara F, et al. LNGFR(+)THY-1(+)VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Rep. 2013;1:152–65.

    Article  CAS  Google Scholar 

  49. Ogata Y, Mabuchi Y, Yoshida M, Suto EG, Suzuki N, Muneta T, et al. Purified human synovium mesenchymal stem cells as a good resource for cartilage regeneration. PLoS One. 2015;10:e0129096.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Yasui T, Mabuchi Y, Toriumi H, Ebine T, Niibe K, Houlihan DD, et al. Purified human dental pulp stem cells promote osteogenic regeneration. J Dent Res. 2016;95:206–14.

    Article  CAS  PubMed  Google Scholar 

  51. Janebodin K, Horst OV, Ieronimakis N, Balasundaram G, Reesukumal K, Pratumvinit B, et al. Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice. PLoS One. 2011;6:e27526.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Morikawa S, Mabuchi Y, Niibe K, Suzuki S, Nagoshi N, Sunabori T, et al. Development of mesenchymal stem cells partially originate from the neural crest. Biochem Biophys Res Commun. 2009;379:1114–9.

    Article  CAS  PubMed  Google Scholar 

  53. Bhatt S, Diaz R, Trainor PA: signals and switches in mammalian neural crest cell differentiation. Cold Spring Harb Perspect Biol. 2013;5. doi:10.1101/cshperspect.a008326.

  54. Langenbach F, Handschel J. Effects of dexamethasone, ascorbic acid and beta-glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res Ther. 2013;4:117.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Liu HC, E LL, Wang DS, Su F, Wu X, Shi ZP, et al. Reconstruction of alveolar bone defects using bone morphogenetic protein 2 mediated rabbit dental pulp stem cells seeded on nano-hydroxyapatite/collagen/poly(L-lactide). Tissue Eng Part A. 2011;17:2417–33.

    Article  CAS  PubMed  Google Scholar 

  56. Yamada Y, Nakamura S, Ito K, Sugito T, Yoshimi R, Nagasaka T, et al. A feasibility of useful cell-based therapy by bone regeneration with deciduous tooth stem cells, dental pulp stem cells, or bone-marrow-derived mesenchymal stem cells for clinical study using tissue engineering technology. Tissue Eng Part A. 2010;16:1891–900.

    Article  CAS  PubMed  Google Scholar 

  57. Wang X, Sha XJ, Li GH, Yang FS, Ji K, Wen LY, et al. Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol. 2012;57:1231–40.

    Article  CAS  PubMed  Google Scholar 

  58. Hosoya A, Hiraga T, Ninomiya T, Yukita A, Yoshiba K, Yoshiba N, et al. Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells. Histochem Cell Biol. 2012;137:733–42.

    Article  CAS  PubMed  Google Scholar 

  59. Chung MT, Liu C, Hyun JS, Lo DD, Montoro DT, Hasegawa M, et al. CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells. Tissue Eng Part A. 2013;19:989–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kim YK, Nakata H, Yamamoto M, Miyasaka M, Kasugai S, Kuroda S. Osteogenic potential of mouse periosteum-derived cells sorted for CD90 in vitro and in vivo. Stem Cells Transl Med. 2016;5:227–34.

    Article  CAS  PubMed  Google Scholar 

  61. Nakamura H, Yukita A, Ninomiya T, Hosoya A, Hiraga T, Ozawa H. Localization of Thy-1-positive cells in the perichondrium during endochondral ossification. J Histochem Cytochem. 2010;58:455–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Bronckaers A, Hilkens P, Fanton Y, Struys T, Gervois P, Politis C, et al. Angiogenic properties of human dental pulp stem cells. PLoS ONE. 2013;8:e71104.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Nakashima M, Iohara K, Sugiyama M. Human dental pulp stem cells with highly angiogenic and neurogenic potential for possible use in pulp regeneration. Cytokine Growth Factor Rev. 2009;20:435–40.

    Article  CAS  PubMed  Google Scholar 

  64. Aranha AM, Zhang Z, Neiva KG, Costa CA, Hebling J, Nor JE. Hypoxia enhances the angiogenic potential of human dental pulp cells. J Endod. 2010;36:1633–7.

    Article  PubMed  Google Scholar 

  65. Matsushita K, Motani R, Sakuta T, Yamaguchi N, Koga T, Matsuo K, et al. The role of vascular endothelial growth factor in human dental pulp cells: induction of chemotaxis, proliferation, and differentiation and activation of the AP-1-dependent signaling pathway. J Dent Res. 2000;79:1596–603.

    Article  CAS  PubMed  Google Scholar 

  66. Tran-Hung L, Laurent P, Camps J, About I. Quantification of angiogenic growth factors released by human dental cells after injury. Arch Oral Biol. 2008;53:9–13.

    Article  CAS  PubMed  Google Scholar 

  67. Tran-Hung L, Mathieu S, About I. Role of human pulp fibroblasts in angiogenesis. J Dent Res. 2006;85:819–23.

    Article  CAS  PubMed  Google Scholar 

  68. Hilkens P, Fanton Y, Martens W, Gervois P, Struys T, Politis C, et al. Pro-angiogenic impact of dental stem cells in vitro and in vivo. Stem Cell Res. 2014;12:778–90.

    Article  CAS  PubMed  Google Scholar 

  69. Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation. 2005;80:836–42.

    Article  PubMed  Google Scholar 

  70. Zhao Y, Wang L, Jin Y, Shi S. Fas ligand regulates the immunomodulatory properties of dental pulp stem cells. J Dent Res. 2012;91:948–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Yamaza T, Kentaro A, Chen C, Liu Y, Shi Y, Gronthos S, et al. Immunomodulatory properties of stem cells from human exfoliated deciduous teeth. Stem Cell Res Ther. 2010;1:5.

    Article  PubMed  PubMed Central  Google Scholar 

  72. Omi M, Hata M, Nakamura N, Miyabe M, Kobayashi Y, Kamiya H, et al. Transplantation of dental pulp stem cells suppressed inflammation in sciatic nerves by promoting macrophage polarization towards anti-inflammation phenotypes and ameliorated diabetic polyneuropathy. J Diabetes Investig. 2016;7:485–96.

    Article  CAS  PubMed  Google Scholar 

  73. Sakai K, Yamamoto A, Matsubara K, Nakamura S, Naruse M, Yamagata M, et al. Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms. J Clin Invest. 2012;122:80–90.

    CAS  PubMed  Google Scholar 

  74. Demircan PC, Sariboyaci AE, Unal ZS, Gacar G, Subasi C, Karaoz E. Immunoregulatory effects of human dental pulp-derived stem cells on T cells: comparison of transwell co-culture and mixed lymphocyte reaction systems. Cytotherapy. 2011;13:1205–20.

    Article  CAS  PubMed  Google Scholar 

  75. Yamaguchi S, Shibata R, Yamamoto N, Nishikawa M, Hibi H, Tanigawa T, et al. Dental pulp-derived stem cell conditioned medium reduces cardiac injury following ischemia-reperfusion. Sci Rep. 2015;5:16295.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Matsubara K, Matsushita Y, Sakai K, Kano F, Kondo M, Noda M, et al. Secreted ectodomain of sialic acid-binding Ig-like lectin-9 and monocyte chemoattractant protein-1 promote recovery after rat spinal cord injury by altering macrophage polarity. J Neurosci. 2015;35:2452–64.

    Article  PubMed  Google Scholar 

  77. Annibali S, Cicconetti A, Cristalli MP, Giordano G, Trisi P, Pilloni A, et al. A comparative morphometric analysis of biodegradable scaffolds as carriers for dental pulp and periosteal stem cells in a model of bone regeneration. J Craniofac Surg. 2013;24:866–71.

    Article  PubMed  Google Scholar 

  78. de Mendonca Costa A, Bueno DF, Martins MT, Kerkis I, Kerkis A, Fanganiello RD, et al. Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells. J Craniofac Surg. 2008;19:204–10.

    Article  PubMed  Google Scholar 

  79. Yamada Y, Ito K, Nakamura S, Ueda M, Nagasaka T. Promising cell-based therapy for bone regeneration using stem cells from deciduous teeth, dental pulp, and bone marrow. Cell Transplant. 2011;20:1003–13.

    Article  PubMed  Google Scholar 

  80. Ito K, Yamada Y, Nakamura S, Ueda M. Osteogenic potential of effective bone engineering using dental pulp stem cells, bone marrow stem cells, and periosteal cells for osseointegration of dental implants. Int J Oral Maxillofac Implants. 2011;26:947–54.

    PubMed  Google Scholar 

  81. Alkaisi A, Ismail AR, Mutum SS, Ahmad ZA, Masudi S, Abd Razak NH. Transplantation of human dental pulp stem cells: enhance bone consolidation in mandibular distraction osteogenesis. J Oral Maxillofac Surg. 2013;71(10):1758.e1–13.

  82. Zheng Y, Liu Y, Zhang CM, Zhang HY, Li WH, Shi S, et al. Stem cells from deciduous tooth repair mandibular defect in swine. J Dent Res. 2009;88:249–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. d’Aquino R, De Rosa A, Lanza V, Tirino V, Laino L, Graziano A, et al. Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge biocomplexes. Eur Cell Mater. 2009;18:75–83.

    Article  PubMed  Google Scholar 

  84. Pisciotta A, Riccio M, Carnevale G, Beretti F, Gibellini L, Maraldi T, et al. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo. PLoS One. 2012;7:e50542.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Giuliani A, Manescu A, Langer M, Rustichelli F, Desiderio V, Paino F, et al. Three years after transplants in human mandibles, histological and in-line holotomography revealed that stem cells regenerated a compact rather than a spongy bone: biological and clinical implications. Stem Cells Transl Med. 2013;2:316–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Riccio M, Maraldi T, Pisciotta A, La Sala GB, Ferrari A, Bruzzesi G, et al. Fibroin scaffold repairs critical-size bone defects in vivo supported by human amniotic fluid and dental pulp stem cells. Tissue Eng Part A. 2012;18:1006–13.

    Article  CAS  PubMed  Google Scholar 

  87. Riccio M, Resca E, Maraldi T, Pisciotta A, Ferrari A, Bruzzesi G, et al. Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures. Eur J Histochem. 2010;54:e46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank our colleagues, laboratory members, and collaborators for their excellent experimental assistance and discussions.

Funding

This work was supported by Japanese Society for the Promotion of Science (JSPS) KAKENHI grants (Grant Nos. 23792293, 25861895, and 16 K11656) to T.Y.

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H.O. is the Editor-in-Chief of this journal and a member of the Scientific Advisory Board of SanBio Co., Ltd (Tokyo, Japan). Y. Matsuzaki concurrently is a director of PuREC, Co., Ltd (Shimane, Japan). The other authors declare that they have no competing of interest.

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Yasui, T., Mabuchi, Y., Morikawa, S. et al. Isolation of dental pulp stem cells with high osteogenic potential. Inflamm Regener 37, 8 (2017). https://doi.org/10.1186/s41232-017-0039-4

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