Open Access

Isolation of dental pulp stem cells with high osteogenic potential

  • Takazumi Yasui1, 2, 3,
  • Yo Mabuchi2, 4,
  • Satoru Morikawa1,
  • Katsuhiro Onizawa3,
  • Chihiro Akazawa4,
  • Taneaki Nakagawa1,
  • Hideyuki Okano2 and
  • Yumi Matsuzaki2, 5Email author
Inflammation and Regeneration201737:8

https://doi.org/10.1186/s41232-017-0039-4

Received: 26 December 2016

Accepted: 23 February 2017

Published: 10 April 2017

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.

Keywords

Bone regenerationDental pulp stem/progenitor cellFlow cytometryIsolationOsteogenic potentialLow-affinity nerve growth factor receptorTHY-1TransplantationCranio-maxillofacial

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 [35]. In particular, DPSCs and SHEDs have a high mineralization potential and are considered to be useful in bone regenerative therapy [68]. 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 [1618]. 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, 4, 2123].

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

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)

Authors

Year

Cell source

Enzyme digestion

Selection

Differentiation

Result

Shi et al. [24]

2003

Human DPSCs

3 mg/ml collagenase type I, 4 mg/ml dispase

STRO-1+ (MACS)

Odontogenic/osteogenic cells

Production of osteodentin-like structures and fibrous connective tissues

Laino et al. [27]

2006

Human DPSCs/SHEDs

3 mg/ml collagenase type I, 4 mg/ml dispase

c-Kit+CD34+STRO-1+CD45 (FACS)

Osteogenic cells

High positivity for CD44, RUNX2, and osteocalcin

Iohara et al. [31]

2006

Human, bovine, canine, and porcine DPSCs

Hoechst 33342 (FACS)

Odontogenic, chondrogenic, adipogenic, and neurogenic cells

SP cells are enriched for stem cell properties and useful for cell therapy with BMP2 to regenerate dentin

Yang et al. [25]

2007

Rat DPSCs

STRO-1+ (FACS)

Odontogenic, neurogenic, adipogenic, myogenic, and chondrogenic cells

STRO-1 selection obtains a more homogeneous cell population with a multilineage differentiation capacity

Honda et al. [30]

2007

Human DPSCs

Hoechst 33342 (FACS)

Odontogenic cells

SP cells expressing ABCG2 in human adult dental pulp that differentiate into odontoblast-like cells

Waddington et al. [39]

2009

Rat DPSCs

4 mg/ml collagenase, 4 mg/ml dispase

LNGFR (MACS)

Osteogenic, adipogenic, \and chondrogenic cells

LNGFR+ DPSCs express CD105 and Notch 2

Ricco et al. [87]

2010

Human DPSCs

3 mg/ml collagenase type I, 4 mg/ml dispase

CD34+c-Kit+STRO-1+ (MACS)

Osteogenic cells

CD34+c-Kit+STRO-1+ DPSCs produce mineralized matrix in 2D and 3D cultures

Iohara et al. [33]

2011

Dog DPSCs

CD105 (FACS)

Odontogenic/osteogenic, adipogenic, angiogenic, and neurogenic cells

Transplantation of CD105+ DPSCs with SDF-1 completely regenerates dental pulp in a pulpectomy model

Mikami et al. [38]

2011

Human SHEDs

2 mg/ml collagenase type I

CD271+CD90+CD44 (FACS)

Osteogenic, adipogenic, chondrogenic, and myogenic cells

LNGFR positivity inhibits the differentiation of DPSCs into osteogenic, adipogenic, chondrogenic, and myogenic lineages

Hosoya et al. [58]

2012

Rat DPSCs

2 mg/ml collagenase, 0.25% trypsin

THY-1High+ (FACS)

Osteogenic cells

Hard tissue formation upon subcutaneous transplantation of THY-1+ cells

Kawanabe et al. [37]

2015

Human SHEDs

5 mg/ml collagenase type II, 2.5 mg/ml dispase I

SSEA-4+ (FACS)

Osteogenic, adipogenic, and chondrogenic cells

SSEA-4+ SHEDs have a multilineage potential.

Yasui et al. [50]

2016

Human DPSCs

2 mg/ml collagenase, 4 mg/ml dispase

LNGFRLow+THY-1High+ (FACS)

Osteogenic, adipogenic, and chondrogenic cells

LNGFRLow+THY-1High+ DPSCs promote osteogenic differentiation.

BMP2 bone morphogenetic protein 2, FACS fluorescence-activated cell sorting, LNGFR low-affinity nerve growth factor, MACS magnetic-activated cell sorting, SDF-1 stromal cell-derived factor-1, SP side population, SSEA-4 stage-specific embryonic antigen-4, THY-1 thymocyte antigen 1

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, 2426] 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, CD31CD146 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 CD31CD146 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+, LNGFRTHY-1Low+, LNGFR+THY-1, and LNGFRTHY-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 [35]. 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 [6268]. Furthermore, DPSCs and SHEDs may have stronger immunomodulatory properties and high anti-apoptotic activity [6976]. Thus, DPSCs and SHEDs could also have potential for clinical applications in autologous stem cell transplantation for bone regenerative therapy.
Fig. 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

Authors and year

 

Targeted site

Cell source

Selection

Host

Scaffolds

Results

de Mendonca et al. [78]

2008

Cranial bone defect

Human DPSCs

Rat

Collagen membrane

Induction of mature bone formation

Seo et al. [8]

2008

Critical-size calvarial bone defect

Human SHEDs

Mouse

HA/TCP

Repair of defects and substantial bone formation

Zheng et al. [82]

2009

Orofacial bone defects

Stem cells from porcine (miniature pig) deciduous teeth

Miniature pig

β-TCP

More efficient regeneration of critical-size mandibular bone defects

d’Aquino et al. [83]

2009

Alveolar bone defect after extraction of impacted third molars

Human DPSCs

Human

Collagen sponge

Complete restoration of bone defects

Ito et al. [80]

2011

Osseointegration of dental implants

Canine DPSCs

Dog

PRP

High osteogenic potential to assist dental implant integration

Yamada et al. [79]

2011

Mandibular bone defect

Canine DPSCs and stem cells from deciduous teeth

Dog

PRP

Well-formed mature bone using both cell lines

Liu et al. [55]

2011

Critical-size alveolar bone defect

Rabbit DPSCs

Rabbit

rhBMP2 + nHAC/PL

Early mineralization and excellent bone formation

Ricco et al. [86]

2012

Critical-size cranial bone defect

Human DPSCs

CD34+c-Kit+STRO-1+ (MACS)

Rat

Fibroin scaffolds

Mature bone formation and defect correction

Pisciotta et al. [84]

2012

Critical-size parietal bone defect

Human DPSCs

STRO-1+ (MACS)

Rat

Collagen constructs

Restoration of critical parietal bone defects

Alkaisi et al. [81]

2013

Distracted area of mandibular bone

Human SHEDs

Rabbit

Enhancement of the bone consolidation period in mandibular distraction osteogenesis

Annibali et al. [77]

2013

Critical-size calvarial bone defect

Human DPSCs/PeSCs

Mouse

Porcine collagen + GDPB, β-TCP, Aga/nHA

β-TCP alone is more effective than β-TCP seeded with DPSCs/PeSCs

Giuliani et al. [85]

2013

Mandibular bone defect after tooth extraction

Human DPSCs

CD34+ (MACS)

Human

Collagen sponge

Regeneration of compact-type bone with uniform vascularization

Yasui et al. [50]

2016

Critical-size calvarial bone defect

Human DPSCs

LNGFRLow+THY-1High+ (FACS)

Mouse

Collagen membrane

LNGFRLow+/THY-1High+ DPSCs promote new bone formation to repair critical-size calvarial defects

Aga/nHA a sponge of agarose and nanohydroxyapatite, DPSCs dental pulp stem/progenitor cells, FACS fluorescence-activated cell sorting, GDPB granular deproteinized bovine bone, HA hydroxyapatite, LNGFR low-affinity nerve growth factor, MACS magnetic-activated cell sorting, nHAC/PLA nanohydroxyapatite/collagen/poly(L-lactide), PeSCs periosteal stem cells, PRP platelet-rich plasma, rhBMP-2 recombinant human bone morphogenetic protein 2, SHEDs stem cells from human exfoliated deciduous teeth, TCP tricalcium phosphate, THY-1 thymocyte antigen 1

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

Declarations

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.

Availability of data and materials

Not applicable.

Authors’ contributions

All authors have read and approved the final manuscript.

Competing interests

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.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Dentistry and Oral Surgery, Keio University School of Medicine
(2)
Department of Physiology, Keio University School of Medicine
(3)
Department of Dentistry and Oral Surgery, Kawasaki Municipal Kawasaki Hospital
(4)
Department of Biochemistry and Biophysics, Graduate School of Health Care Sciences, Tokyo Medical and Dental University
(5)
Department of Cancer Biology, Faculty of Medicine, Shimane University

References

  1. Liu H, Gronthos S, Shi S. Dental pulp stem cells. Methods Enzymol. 2006;419:99–113.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.PubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle Scholar
  11. Cannon TY, Strub GM, Yawn RJ, Day TA. Oromandibular reconstruction. Clin Anat. 2012;25:108–19.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.PubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.PubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.PubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticleGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle Scholar
  67. Tran-Hung L, Mathieu S, About I. Role of human pulp fibroblasts in angiogenesis. J Dent Res. 2006;85:819–23.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.PubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedGoogle 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.PubMedGoogle 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.Google Scholar
  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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedPubMed CentralGoogle 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.View ArticlePubMedGoogle 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.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© The Author(s) 2017