osteoblastic differentiation of dental pulp cells

archives of oral biology 59 (2014) 310–317

Available online at www.sciencedirect.com

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Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells Mioko Yamamoto a, Nobuyuki Kawashima a,*, Nami Takashino a, Yu Koizumi a, Koyo Takimoto a, Noriyuki Suzuki a, Masahiro Saito b, Hideaki Suda a a

Pulp Biology and Endodontics, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan b Division of Operative Dentistry, Department of Restorative Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan

article info

abstract

Article history:

Objective: Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations

Accepted 18 December 2013

of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of

Keywords:

immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into

Spheroid culture

odontoblasts.

Dental papilla cells

Methods: We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates

Odonto/osteoblastic differentiation

for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture.

Integrin signalling

Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signallingrelated proteins were detected immunohistochemically. Results: Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs ( p < 0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. Conclusion: 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling. # 2013 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +81 3 5803 5495; fax: +81 3 5803 5494. E-mail address: [email protected] (N. Kawashima). Abbreviations: 2D, two-dimensional; 3D, three-dimensional; ALP, alkaline phosphatase; a-MEM, alpha-modified minimum essential medium; BMP, bone morphogenetic protein; DMP1, dentine matrix protein-1; Dspp, dentine sialophosphoprotein; ECM, extracellular matrix; FAK, focal adhesion kinase; FBS, foetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MSCs, mesenchymal stem cells; Oc, osteocalcin; pFAK, phosphorylated focal adhesion kinase; pPaxillin, phosphorylated paxillin; PLGA, poly (lactic-co-glycolic acid); qPCR, quantitative PCR. 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.006

archives of oral biology 59 (2014) 310–317

1.

Introduction

Odontoblasts arise from mesenchymal dental papilla cells, and their differentiation is guided by molecular crosstalk between the ectoderm and mesenchyme from the cap stage of tooth development. Various growth factors, such as bone morphogenetic protein (BMP)-2,1,2 BMP-4,3,4 BMP-7,5 epidermal growth factor6,7 and fibroblast growth factors,8 are thought to be involved in odontoblastic differentiation of dental papilla/pulp cells. Odontoblastic differentiation is also induced by conditioned medium of cultured tooth germ cells, suggesting that various growth factors secreted from tooth germ cells are essential for odontoblastic differentiation.9,10 To induce odontoblastic differentiation of dental pulp cells, most experiments are performed in culture dishes or wells. However, tissues are three-dimensional (3D) and organized by complicated cell–cell and cell–extracellular matrix (ECM) interactions. Therefore, traditional 2D monolayer culture systems have a limitation to reproduce the events occurring in normal cell physiology. To overcome this limitation, various methods have been investigated for cell culture in 3D environments.11–14 3D culture systems either use scaffold materials or are ‘‘scaffold-free’’. The former culture system is relatively popular, because it is possible to change the cell density and shape of samples easily and differentiation can be directed by the nature of scaffold materials and addition of growth factors. Collagen,15 gelatin,16 and poly (lactic-co-glycolic acid) (PLGA)17 are often used in scaffolds and act as a frame to guide cultured cells. However, it has been reported that scaffold materials can cause infection and/or inflammation during degradation in vivo.18 In addition, cells are surrounded by artificial materials in scaffolds and the environment is different from the in vivo cell–cell and cell–ECM attachments in the tissue. 3D spheroid culture is a method for creating 3D aggregations of cells and their ECM without a scaffold,19,20 which provides physiological cell culture conditions that mimic the actual tissues. Such systems have been applied to studies of tumours,21 cell–cell interactions,22 tissue engineering,23–28 embryonic/induced pluripotent stem cell and mesenchymal stem cell (MSC) differentiation,14,29,30 and cell death.31 However, there are only few reports on 3D spheroid culture systems in dental pulp research. Iohara et al. reported that porcine primary pulp cells differentiate into odontoblasts effectively by pellet culture, one of the 3D spheroid culture systems.23 Nonetheless, little is known about the precise effects of 3D spheroid culture on the properties of dental pulp cells. In this study, immortalized mouse dental papilla cells (MDPs), which possess dental pulp cell properties and the ability to differentiate into odontoblasts,32 were cultured in a 3D spheroid culture system to evaluate their phenotypic changes.

2.

Materials and methods

2.1.

Cell culture

We cultured MDPs in alpha-modified minimum essential medium (a-MEM, Wako Pure Chemical Industries, Osaka, Japan) containing 10% foetal bovine serum (FBS, Thermo

311

Fisher Scientific, Waltham, MA, USA) and an antibiotic and anti-fungus solution (Penicillin–Streptomycin–Amphotericin B suspension; Wako Pure Chemical Industries) at 37 8C/5% CO2. Cells were cultured for 0 (control), 1, 3, 7, and 14 days in 96-well low-attachment culture plates (PrimeSurface; Sumitomo Bakelite, Tokyo, Japan) at a seeding density of 3  104 cells/well for 3D spheroid culture. We used flatbottomed culture plates for 2D monolayer culture, and the same density of cells. The control cells at day 0 were an aliquot of MDPs prior to seeding. Medium was changed every 3 days. The diameter of each spheroid colony was measured under a microscope (Carl Zeiss, Oberkochen, Germany). For mineralized nodule formation, MDPs were cultured in 96-well lowattachment culture plates at a seeding density of 3  104 cells/ well for 3D spheroid culture, or in 48-well flat-bottomed culture plates for 2D monolayer culture at a seeding density of 5  104 cells/well for 3 days. Then, the medium was changed to mineralization-inducing medium containing 0.2 mM L-ascorbic acid phosphate magnesium salt (Wako Pure Chemical Industries) and 5 mM b-glycerophosphoric acid (Sigma, St. Louis, MO, USA). The cells were then cultured for another 2 or 6 days.

2.2.

Analysis of hypoxia

The levels of hypoxia were determined by the hypoxia probe LOX-1 (50 mM; SCIVAX, Kanagawa, Japan). LOX-1 is a phosphorescent light-emitting iridium complex that is quenched by oxygen, and its phosphoresce decreases in response to the level of oxygen.33 LOX-1 was added to the culture medium at 24 h before detection. 2D monolayer-cultured MDPs in a low O2 atmosphere (5%) were used as a positive control, and cells cultured in a normal O2 atmosphere (20%) were used as a negative control.

2.3.

Histology and immunohistochemistry

We fixed MDPs cultured on a plastic film (Cell Desk LF; Sumitomo Bakelite) with 4% paraformaldehyde for 15 min at 4 8C. Spheroid colonies of MDPs were fixed for 30 min at 4 8C and then embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and frozen quickly in dry ice/hexane. The 2D monolayer-cultured MDPs and cryostat sections (7 mm) of spheroid colonies were subjected to haematoxylin and eosin (HE) staining, alkaline phosphatase (ALP) staining, and immunohistochemistry. A mixture of naphthol AS-MX sodium salt (Sigma) and Fast-Blue RR salt (Sigma) was used for ALP staining. For immunohistochemistry, the primary antibodies were anti-Ki67 (1:1000; Thermo Fisher Scientific), anti-cleaved caspase-3 (1:3000; Cell Signaling Technology, Danvers, MA, USA), anti-dentine matrix protein-1 (DMP1, 1:1000; Takara Bio, Otsu, Japan), anti-vinculin (1:20,000; Sigma), anti-phosphorylated focal adhesion kinase (Tyr576/ 577) (pFAK, 1:2000; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-phosphorylated paxillin (Tyr31) (pPaxillin, 1:500; GeneTex, Irvine, CA, USA). Immunoreactivity was detected by the ABC method (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) and the colour reaction was developed with 3,30 -diaminobenzidin (Vector Laboratories). For mineralized nodule detection, MDPs were stained

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Table 1 – Primer sequences. Genes

Upper primers 0

Lower primers 0

with an Alizarin red S solution (40 mM Alizarin Red-S, pH 4.2, [Sigma]) after methanol fixation.

RNA extraction and quantitative PCR (qPCR)

Total RNA (300 ng) extracted from 2D monolayer- or 3D spheroid-cultured MDPs using an RNAqueous-Micro kit (Life Technologies, Carlsbad, CA, USA) was converted into cDNA using RevertAid H Minus M-MuLV Reverse Transcriptase (Thermo Fisher Scientific), an oligo dT primer (Life technologies), and dNTP Mix (Life Technologies). The qPCR assays were conducted with cDNA (0.8 ml) using specific primers (Table 1), CFX96 (Bio-Rad, Hercules, CA, USA), and GoTaq qPCR Master Mix (Promega, Madison, WI, USA) in a total volume of 10 ml. Gene expression was normalized to that of the internal control (b-actin). Five samples were analyzed for each group. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also used as an internal control, but there were no differences between data obtained using b-actin and GAPDH (data not shown).

2.5.

5 -AACCGCTCGTTGCCAA-30 50 -CGTCCTTGTCCTCACTCTCG-30 50 -TGGTCACTATTTGCCTGTGC-30 50 -GCCCTCTCCAAGACATATA-30 50 -AAATAGTGATACCGTAGATGCG-30 50 -GCTGTTTGTGTTTGGCTTGA-30

5 -AGGGAAATCGTGCGTG-3 50 -CACATGCGGGAGAAGACG-30 50 TCTCCCAGTTGCCAGATACC-30 50 -CCATGATCACGTCGATATCC-30 50 -TCTGACAAAGCCTTCATGTCC-30 50 -TGGAAGTGGCCCATTTAGAG-30

b-Actin Dspp Dmp1 Alp Oc Bmp2

2.4.

0

Statistical analysis

Differences between groups were evaluated by the Kruskal– Wallis test and Scheffe´’s method. A p value less than 0.05 were considered statistically significant.

3.

Results

3.1.

Morphology

MDPs cultured in a 96-well low-attachment plate formed spherical colonies from day 1. The spheroid colonies were translucent and concave at day 1; by day 14, colonies were opaque white and completely spherical (Fig. 1A). The diameters of spheroid colonies were 750.8  23.8 mm, 706.3  25.0 mm, and 919.5  33.8 mm at days 1, 3, and 14, respectively (Fig. 1B). 2D monolayer-cultured MDPs were spindle shaped (Fig. 2A), and 3D spheroid-cultured MDPs were a cuboidal shape (Fig. 2B–E). 3D spheroid-cultured MDPs aggregated loosely and were distributed homogeneously at day 1 (Fig. 2B). During culture, dense cell distribution was observed at the periphery of the spheroid colonies, with the centres of the sphere lacking large numbers of cells (Fig. 2C–E). Ki67-positive proliferating MDPs were mainly found in the periphery of spheroid colonies, and their numbers decreased over time (Fig. 2F–I). Cleaved caspase-3-positive apoptotic MDPs were detected in the core zone, and their number increased over time (Fig. 2J–M).

3.2.

Hypoxia

2D monolayer-cultured MDPs in 20% O2, which were used as a negative control, showed few LOX-1-positive hypoxic cells (Fig. 3A), whereas MDPs cultured in 5% O2, which were used as a positive control, showed many hypoxic cells (Fig. 3B). A hypoxic condition was detected by LOX-1 in the spheroid colonies of MDPs at day 3, especially in the core zone of spheroids (Fig. 3C).

3.3. Odonto/osteoblastic differentiation marker gene expression Odonto/osteoblastic differentiation marker expression was evaluated in 2D monolayer- and 3D spheroid-cultured MDPs by qPCR. The 2D monolayer-cultured MDPs showed low expression levels of dentine sialophosphoprotein (Dspp), Dmp1, Alp, osteocalcin (Oc), and Bmp2. These expression levels did not change during the course of the cell culture period (data not shown). Expression of Dspp (Fig. 4A) and Dmp1 (Fig. 4B) was significantly increased in 3D spheroid-cultured MDPs at day 14 ( p < 0.05). Expression of Alp was significantly increased in 3D spheroid-cultured MDPs at day 1, and continued to increase until day 14 ( p < 0.05; Fig. 4C). Expression of Bmp2 was significantly increased in 3D spheroid-cultured MDPs at day 1, with a further increase observed at day 7 ( p < 0.05; Fig. 4D).

Fig. 1 – Increase in the diameter of 3D spheroid colonies of MDPs. Photograph of 3D spheroid colonies (A). Scale bar: 1 mm. Change in diameter of 3D spheroid colonies of MDPs during culture (B). ( p < 0.05 vs. the minimum diameter of spheroid colonies at day 3, n = 8).

archives of oral biology 59 (2014) 310–317

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Fig. 2 – Morphology of 2D monolayer- and 3D spheroid-cultured MDPs. HE staining (A–E) revealed that 2D monolayercultured MDPs had a spindle shape (A). Cuboidal 3D spheroid-cultured MDPs were scattered at day 1 (B), however dense cell distribution in the peripheral zone was observed during culture (C: day 3, D: day 7, E: day 14). Ki67-positive proliferative cells were found mainly in the periphery of spheroid colonies (F: day 1, G: day 3, H: day 7, I: day 14). Expression of cleaved caspase-3 was mainly detected in the core zone (J: day 1, K: day 3, L: day 7, M: day 14). Lower panels: high-power views of boxed areas in upper panels. Scale bars: 50 mm.

Expression of Oc was significantly increased at day 7, and a further increase was observed at day 14 ( p < 0.05; Fig. 4E).

3.4. ALP activity, DMP1 expression, and mineralized nodule formation Low ALP activity was detected in 2D monolayer-cultured MDPs (Fig. 5A), which did not change during long-term culture (data

not shown). 3D spheroid-cultured MDPs showed low ALP activity at day 1 (Fig. 5B), which increased during culture. Higher ALP activity was detected in the periphery of spheroid colonies (Fig. 5C–E). Low expression of DMP1 was observed in 2D monolayer-cultured (Fig. 6A), but 3D spheroid-cultured MDPs showed relatively high expression of DMP1 at day 1 (Fig. 6B), which increased over the course of the culture period (Fig. 6C). DMP-1 expression was high and low in the periphery

Fig. 3 – Hypoxic condition of the spheroid colonies. Few hypoxic cells (arrows) were observed among 2D monolayer-cultured MDPs at a normal concentration of O2 (A), but most cells showed hypoxia (arrows) among 2D monolayer-cultured MDPs at 5% O2 (B). The centre of spheroid colonies became strongly hypoxic at day 3 in 20% O2 (C). Dotted outline indicates a spheroid colony. Scale bars: 50 mm.

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Fig. 4 – Odonto/osteoblastic marker gene expression of 3D spheroid-cultured MDPs. Expression of Dspp (A), Dmp1 (B), Alp (C), Bmp2 (D), and Oc (E) was significantly up-regulated in 3D spheroid-cultured MDPs. (*p < 0.05 vs. control, #p < 0.05 vs. expression levels of the group from the previous period, n = 5).

and centre of spheroid colonies, respectively. Few mineralized nodules were observed in 2D monolayer-cultured MDPs in the presence of mineralization-inducing medium at day 2 (Fig. 7A), but a relatively large number of mineralized nodules were observed at day 6 (Fig. 7B). In contrast, a high rate of mineralized nodule formation was observed in 3D spheroidcultured MDPs in the presence of mineralization-inducing medium at day 2 (Fig. 7C), which was enhanced at day 6 (Fig. 7D).

3.5.

Expression of integrin signalling-related proteins

Vinculin, pFAK, and pPaxillin expression was low in the 2D monolayer-cultured MDPs, whilst relatively strong expression of these molecules was detected in spheroid colonies at day 14, particularly in the peripheral zone (Fig. 8).

4.

Discussion

We induced odonto/osteoblastic differentiation of MDPs cultured under a 3D spheroid condition without any specific differentiation-inducing factors such as BMPs and transforming growth factor-b. On the other hand, 2D monolayercultured MDPs in conventional medium did not undergo

odonto/osteoblastic differentiation. MDPs were derived from the dental papilla cells of ICR mouse incisors, and immortalized by infection with the mutant human papilloma virus 16E6 delta146-151 that specifically inactivates p53.32 MDPs possess dental pulp cell properties and the ability to differentiate into odontoblasts.32 We revealed that 3D spheroid-cultured MDPs showed significant up-regulation of odonto/osteoblastic differentiation marker gene expression (Dspp, Dmp1, Alp, Oc, and Bmp2), ALP activity, and DMP1 expression compared with that in 2D monolayer-cultured cells without any induction stimuli. Furthermore, the 3D spheroid culture system enhanced mineralized nodule formation in the presence of mineralization-inducing medium compared with that in the 2D monolayer culture system. The environment of the 3D spheroid culture system, which mimics that of tissues, may have an advantage for differentiation of cultured cells. In contrast, 2D monolayer-cultured cell attachment to a plastic surface is artificial, which may suppress their original properties. Pellet culture, which is another method of 3D spheroid culture, also induces odonto/osteoblastic differentiation of primary dental pulp cells23 and MSCs.25,34 Although the mechanism of differentiation induction is unclear, enhanced cell–cell and cell–ECM interactions among 3D spheroid-cultured MDPs, which are suggested by high expression of vinculin,35 may be a potent inducer of odonto/

Fig. 5 – ALP activity of 2D monolayer- and 3D spheroid-cultured MDPs. Higher ALP activity was observed in 3D spheroidcultured MDPs compared with that in 2D monolayer-cultured MDPs (A). At day 1, 3D spheroid-cultured MDPs showed low ALP activity (B), but their ALP activity increased during the culture period (C: day 3, D: day 7, E: day 14). Scale bars: 50 mm.

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Fig. 6 – DMP1 expression of 2D monolayer- and 3D spheroid-cultured MDPs. 2D monolayer-cultured MDPs showed low DMP1 expression (A), but relatively strong expression of DMP1 was observed in 3D spheroid-cultured MDPs at day 1 (B). DMP1 expression of 3D spheroid-cultured MDPs was higher at day 14 (C). Scale bars: 50 mm.

osteoblastic differentiation. A major family of adhesion molecules on the cell surface is the integrins36 that are essential for spheroid formation. Expression of integrins is upregulated in 3D spheroid-cultured cells, and inhibition of such expression disrupts 3D spheroid formation of MG63 cells.37 Integrin signalling also plays important roles in odontoblastic differentiation of neural crest-derived ectomesenchymal cells.38 Promotion of integrin signalling by administration of dentine phophoprotein to undifferentiated dental pulp cells induces expression of DMP1 and DSPP via phosphorylation of paxillin and FAK which are major integrin signalling molecules.39 We revealed up-regulation of pPaxillin and pFAK expression in 3D spheroid-cultured MDPs compared with that in the 2D monolayer-cultured MDPs. We also found up-regulation of Bmp2 expression in 3D spheroid-cultured MDPs. BMP-2 is a potent inducer of odontoblastic differentiation in dental pulp cells,40,41 and

Fig. 7 – Mineralized nodule formation in 2D monolayer- and 3D spheroid-cultured MDPs. Few mineralized nodules were observed in 2D monolayer-cultured MDPs at day 2 (A), and relatively high number of nodules were observed at day 6 (B). 3D spheroid-cultured MDPs showed high mineralized nodule formation at day 2 (C), which increased at day 6 (D). Scale bars: 2 mm (A and B) and 200 mm (C and D).

up-regulation of Dspp expression in MDPs is induced by BMP2.42 In this study, Bmp2 expression increased in 3D spheroid-cultured MDPs until day 7, suggesting that BMP-2 further promoted the odontoblastic differentiation of MDPs, which was evaluated by the increase of Dspp expression. Bmp2 gene expression is also significantly up-regulated in 3D spheroid-cultured human bone marrow MSCs up to day 7.34 Histologically, the 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. The bilayer structure became clearer during the culture period. Pellet-cultured bone marrow-derived MSCs, synovial MSCs, and chondrocytes under cartilage also show a layered structure.41 Nutritional supply and an ideal oxygen concentration in the peripheral area of spheroid colonies are favourable for cell proliferation and differentiation.40 In the present study, odonto/osteoblastic differentiation was induced in spheroid colonies of MDPs, which was confirmed by up-regulation of odonto/osteoblastic markers. In contrast, the core region of spheroid colonies showed a hypoxic condition and the presence of apoptotic cells. Hypoxic

Fig. 8 – Vinculin and integrin signalling-related protein expression of 2D monolayer- and 3D spheroid-cultured MDPs. Vinculin, pFAK, and pPaxillin expression was low in 2D monolayer-cultured MDPs, but strong expression of these proteins was observed in 3D spheroid-cultured MDPs, especially at the periphery of spheroid colonies. Scale bars: 50 mm.

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conditions maintain the stemness of stem cells,43 and the core zone of spheroid colonies may be a niche of stem cells supplying progenitor cells to the differentiating peripheral zone. From a clinical perspective, there are promising applications of 3D spheroid culture systems in dentine and bone regeneration. Clinically, a scaffold is essential for cell adhesion and proliferation, and it provides support and a structure for reconstruction of organs. Biodegradable scaffolds are dissolved and disappear in the body, such as collagen,15 gelatin16 and PLGA,17 and are commonly used for regenerative medicine. However, biodegradable scaffolds are reported to cause strong inflammatory responses through the process of degradation. In particular, the aliphatic polyester PLGA creates an acidic environment that reduces cell viability and migration.44 Polypeptides, such as collagen and gelatin, are hydrolyzed and their biodegradation resembles natural woundhealing responses. However, there are the possibilities of immunogenicity and xenogeneic infection. Scaffold-free transplantation systems, such as 3D spheroid culture systems, can overcome these problems in regenerative medicine. 3D spheroid culture systems include the hanging drop system, spheroid system using low-attachment culture plates, bioreactors, and pellet culture.19 In this study, we used lowattachment culture plates for the spheroid system that allows effective formation of homogeneous spheroid colonies.45 Easy handling and high throughput are also benefits of this system. However, the size of spheroid colonies was relatively small (919.5  33.8 mm in diameter at day 14), and a considerable amount of spheroid colonies may be required for tissue regeneration of large defects. Further studies are necessary for the clinical application of this spheroid system. In conclusion, we found that 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, and is probably mediated by integrin signalling.

Authors’ contribution Yamamoto and Kawashima: study conception and design; Yamamoto, Kawashima, Takashino, Koizumi, Takimoto, and Saito: acquisition of data; Yamamoto: analysis and interpretation of data; Yamamoto: drafting of manuscript; Kawashima and Suda: critical revision.

Funding This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (#25293386 and #25253101 to N.K.).

Competing interests There is no conflict of interest.

Ethical approval Not required.

Acknowledgement We thank Dr. Kayoko Ohnishi for her technical support.

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