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Phenotype properties of a novel spontaneously immortalized odontoblast-lineage cell line
BBRC Biochemical and Biophysical Research Communications 342 (2006) 718–724 www.elsevier.com/locate/ybbrc
Phenotype properties of a novel spontaneously immortalized odontoblast-lineage cell line Szilvia Arany *, Akira Nakata, Takashi Kameda, Souchi Koyota, Yasuharu Ueno, Toshihiro Sugiyama Department of Biochemistry, Akita University School of Medicine, Hondo 1-1-1, Akita 010-8543, Japan Received 1 February 2006 Available online 14 February 2006
Abstract Here we report on the spontaneous immortalization upon serial passages of mouse fetal dental papilla cells, which present odontoblast phenotype features. The cells named odontoblast-lineage cell (OLC) produced dentin extracellular matrix proteins, such as DSP and DMP1, and maintained transcripts of various matrix components as osteopontin, BMP-4, procollagen-1, and MEPE. The addition of osteogenic differentiation medium with b-glycerophosphate and ascorbic acid was effective for inducing calcification and mineralization in vitro in cell cultures for up to 28 days. For the first time, we investigated the expression of Lhx6 and Lhx7 genes during induced biomineralization, since these new members of LIM homeodomain proteins have been recently proposed tracking odontoblastic phenotypes. Our results indicate that b-glycerophosphate treatment of OLC cultures decreases Lhx6 transcript levels in vitro. Our findings proved odontoblast phenotype-specificity, which demonstrates that this novel odontoblast-lineage cell line is a valuable tool for future experiments in odontology. 2006 Elsevier Inc. All rights reserved. Keywords: Odontoblast; Spontaneous immortalization; Dental papilla; Dentin extracellular matrix proteins; Calcification; Lhx6; Lhx7
To create models of dental development and to elucidate epithelial–mesenchymal interactions and pathways of tooth formation, continuous cell lines providing consistent and homogeneous research material are inevitably necessary [1]. These cell lines are considered to be useful tools for tissue engineering purposes, tissue recombinant studies, dental restorative, repairment experiments, as well as, research utilities for dental material testing. Therefore, several attempts focused on the isolation and the production of tooth cells applicable also for cytodifferentiation and biomineralization experiments. Among them, numerous studies shed some light on the dentinogenesis responsible odontoblasts, which are neural crest-derived mesenchymal cells from the first branchial arch. Their differentiation during tooth development and maturation is the result of the signaling cascade between the inner dental epithelium and *
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0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.02.020
the adjacent dental mesenchyme. However, to date, the complexity of the molecular mechanism underlying those interactions is not fully understood [2]. Putative odontoblast progenitor cells have been recognized [3,4] and predetermined odontoblastic population in dental pulp has been described [5] earlier. These systems were principally suitable for the analysis of odontoblast differentiation from tissue explants, although they did not yield continuous cell lines. According to the relevant literature, existing rat dental pulp cell lines; RPC-C2A [6] and RDP 4-1 [7] showed an incomplete odontoblast specific gene expression pattern. A spontaneously transformed mouse dental papilla cell line, called MDPC-23, developed by Hanks et al. [8], has already been chosen for various experiments [9,10] and proven its usefulness and applicability for dental research. It expresses common proteins of mineralizing dentin, as collagen, osteopontin, as well DSP, and inducible to form mineralized nodules in vitro. However, the authors were
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unable to detect transcripts for DMP-1 from MDPC-23 cells, which likely regulates ECM mineralization process in vivo and plays a significant role in terminal differentiation of odontoblasts [11]. Additionally, several authors reported on the advantages of using dental pulp stem cells (DPSC) in topics related to dentinogenesis [12,13]. Those cells obtained from dental pulp can be genetically transformed for odontoblast-like cell required experiments [14]. However, none of those approaches resulted in continuous cell lines from spontaneous immortalization, yet the potential side effects of applied viral oncogenes should be considered. Our laboratory reported on the successful establishment of an ameloblast-lineage cell line (ALC) [15], which represents the epithelial component of tooth germs. As a consecutive part of that study, the present research attempted to develop a continuous cell line from the mesenchymal component of tooth germ, which is devoted to odontoblast development. It has been proposed that a layer of odontoblast-lineage cells is located at the periphery of murine dental papillae [16,17], which encouraged us to employ mouse tooth germ explants as described in details in this paper. Here we present the isolation and the spontaneous immortalization of an odontoblast-lineage cell line, which has been probed to match functional criteria of odontoblasts. Hence, we searched transcripts for some of the small integrin binding ligand N-linked glycoprotein (SIBLING) protein genes, which are believed to play important role in osteogenesis and dentinogenesis [18]. Among the SIBLING proteins, we assayed our cells for DSPP, DMP-1, MEPE, and osteopontin transcripts. We included gene expression experiments on known markers for dentin extracellular matrix (ECM): procollagen-1 (PCOL-1) and BMP-4. Moreover, we tested our established cell line concerning a novel discovery in recognizing odontoblast phenotype. According to a recent advancement, transcripts of Lhx6 and Lhx7 were expressed in dental clones and postulated to be applicable markers of cell lines maintaining properties of odontoblastic phenotype [19]. Lhx6 and Lhx7 are members of a novel subfamily of LIM homeodomain proteins, which have already been detected in the presumptive odontogenic mesenchyme [20]. We performed an experiment to determine whether in vitro, induced calcification with ‘‘osteogenic differentiation medium’’ [21] alters the LIM gene expressions. Materials and methods Cell culture experiments. All experiments on animals were conducted with the approval of the Committee on Animal Experimentation of Akita University. Tooth germs were dissected out from 18.5-day-old C57BL/ 6–TgN (act-EGFP) OsbC14–YO1–FM131 mouse embryos under microscopic observation. Following aseptic isolation, washing with phosphatebuffered saline (PBS), and seeding on type-I collagen coated 60 mm culture dishes (Corning, USA), primary cell cultures were obtained from explants’ outgrowth after 4 weeks. After removing the tooth germs, fibroblastic-like outgrowths were cultured in minimum essential medium alpha modification (a-MEM) (Sigma, USA) supplemented with 15% (v/v) heat-inactivated
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fetal bovine serum (Sigma. USA), and fibroblast growth factor (FGF)-2 (2 ng/ml) on type-I collagen coated 6-well culture plates at 37C in a humified chamber of 5% (v/v) CO2 in air. The spread cells were transferred to new dishes at subconfluence, after 0.05% trypsin–0.02% EDTA digestion and maintained as described previously by Hanks et al. [8]. At passage 15, cells were inoculated at low density (10–15 cells per 60 mm dish) and single cell derived colonies were established for further cultivation. Those subcultures were studied routinely under an inverted microscope. Twenty colonies were picked and investigated for odontoblast phenotype (detailed later in this section). Subsequently, the selected odontoblast-lineage cells (OLCs) were transferred over 60 times to present. RNA preparation, reverse transcriptase-PCR, and quantitative RTPCR. Total RNA was extracted from single cell cloned colonies, likewise OLC cultures after every tenth passage using ISOGEN reagent (Nippon Gene, Japan) according to the manufacturer’s protocol. Concentrations of total RNA were determined by the absorption ratio (OD260/OD280 nm) with UV spectrophotometry (Beckman, Germany). Then the template RNA were processed to evaluate odontoblastic gene expression pattern using the Superscript One-Step RT-PCR with Platinum Taq system (Invitrogen, USA). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. Reaction mixtures that were set up included specific mouse primers sets (10 pM/ll) for dentin sialophosphoprotein (DSP) (5 0 -ACAGCAAATGGCATCAGGAG-3 0 and 5 0 -CT CGTAACTGTACCCGTACT-3 0 ), dentin matrix protein (DMP)-1 (5 0 -GAAAGCTCTGAAGAGAGGAC-3 0 , 5 0 -GTCATCTGAATCCG 0 0 TAGTGC-3 ), procollagen-1 (5 -CTGCCGGAGAAGAAGGAAAA-3 0 , 5 0 -CCATCTTTACCAGGAGAACC-3 0 ), osteopontin 5 0 -TGGACGAC GATGARGACGAT-3 0 , 5 0 -GACTATCGATCACATCCGAC-3 0 ), bone morphogenetic protein (BMP)-4 (5 0 -AGGCGACACTTCTACAGATG3 0 , 5 0 -ATGGCCAAAAGTGACCAGGA-3 0 ), matrix extracellular phosphoglycoprotein (MEPE) (5 0 -GCTCCAGCAAAGCTGAAG-3 0 , 5 0 -AC TAAGACTGTGAGAGGGCA-3 0 ), Lhx6 (5 0 -AATCTATGCCAGTGA CTGGG-3 0 , 5 0 -GTGTCGTAATGGATGCGACA-3 0 ), and Lhx7 0 (5 -TACTTCAGACGGTATGGGAC-3 0 , 5 0 -TCCCATTACCGTTCTCC ACT-3 0 ). We designed the primers using information from GenBank database (http://www.ncbi.nlm.nih.gov/Genbank). The reverse transcriptase PCR conditions were set up as follows: 35 cycles of 60 s denaturation at 94 C, 60 s annealing at 56–58 C, and 90 s extension at 70–72 C. Appropriate negative controls (mouse fibroblast cells for DSP and DMP1) and positive controls (total RNA isolated from mouse tooth germs from post-partum day, ppd1) were included in all examinations. The PCR products were loaded on 1.5% agarose gel for electrophoresis and visualized by ethidium bromide staining under ultraviolet light exposure. The quantification of Lhx6 and Lhx7 transcripts was performed by real-time PCR method using the SuperScript Platinum III Two-Step qRT-PCR Kit with SYBR Green (Invitrogen, USA). For LightCycler experiments (Roche Molecular Biochemicals, Switzerland) 2 ll cDNA template (reverse transcribed total RNA from 2 or 10 mM b-glycerophosphate administered cultures) was added to 18 ll master mix containing the specific primer pairs. The following LightCycler programming was used: initial denaturation (95 C for 2 min), 45 cycles of amplification with a single fluorescent measurement (denaturing at 94 C for 5 s, annealing at 58 C for 10 s, and elongation at 72 C for 10 s), and melting curve analysis with continuous fluorescent measurement. Finally, standard curves from control dilution series were created with LightCycler Software, Version 3.5. The relative quantity of target genes was calculated by the normalization method versus 18S reference gene. Western blotting and immunofluorescence microscopy. Cultured cells at confluence were lysed in PhosphoSafe Extraction Buffer (Novagen, USA) and centrifuged for 5 min at 16,000g at 4 C. The lysate concentration was assayed using the BCA method, and then the protein samples were subjected to SDS–PAGE gel electrophoresis on 10% (w/v) acrylamide gel. Separated proteins were electrophoretically transferred onto PVDF membrane (Immobilion-P, Millipore, USA) with a semi-dry system. After rinsing and blocking (5% dried skim milk) the blots DSP and DMP-1 were detected by commercially available antibodies: by DSP goat polyclonal antibody (Santa Cruz Biotechnology, USA) and by DMP-1 rabbit
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monoclonal antibody (Takara, Japan), respectively. The proteins were visualized by enhanced chemiluminescence (ECL) (Amersham, Sweden) according to the manufacturer’s instructions. For immunohistochemical analysis, cells were cultivated on collagen type-I coated glass coverslips inserted in 24-well plates, and, after reaching confluence, they were fixed in 4% paraformaldehyde. Following permeabilization with 0.1% Triton X-100 in PBS and subsequent blocking, the samples were incubated at 4 C overnight with primary antibodies against DSP and DMP1, diluted 1:300 in blocking solution. Then samples were washed in PBST and incubated with the secondary antibodies; Alexa Fluor 633-conjugated anti-goat antibody (Molecular Probes, USA) and Alexa Fluor 546 anti-rabbit antibody, respectively, diluted 1:500 in PBST. The sections were washed with PBST and mounted onto microscopic slides. Images were obtained and analyzed using the LSM-510 confocal imaging system (Zeiss, Germany). Induction of in vitro mineralization and alizarin red staining. To induce calcification and nodule formation, cells were seeded at a density of 3 · 104 and cultured in conditioned a-MEM medium with 2 mM (low concentration) or 10 mM (high concentration) b-glycerophosphate plus 50 lg/ml ascorbic acid (Sigma, USA) for 4 weeks. The control group was cultured in a-MEM containing 15% FBS. The medium was refreshed every 3 days. On day 28, the cultures were fixed in 4% paraformaldehyde for 10 min and then stained with 1% alizarin red S (Sigma, USA).
Results and discussion Establishment and morphology of cultured mouse tooth germ derived cells To establish immortalized dental cell line with odontoblastic phenotype, tooth germs removed from E18.5 mouse mandibles were placed in culture medium. On the first day,
tooth germs attached to the collagen coated dish, and during the following days outgrowth of dental papilla cells could be observed in most of the dishes. The proliferating cells emerged in a thickening area surrounding the central germ, which was discarded after 1 week. We observed mainly two types of outgrowth, distinguished by cell shape. The spreading cells showed either a polygonal-shaped or a fibroblast-shaped appearance. For further transfer, we selected explants that contained cells exhibiting fibroblastic-like morphology with vigorous outgrowth during 4 weeks. In order to design this experiment, we also referred to a previously described 3T3 regimen [22] of routine subculture. We seeded cells at low density to perform dilution cloning at passage 15. Twenty of the isolated cell populations were collected with cloning rings and allowed to grow to subconfluence. From this point, we continued the regular schedule (serial passage with 3 days interval) on cell transfer and investigated the cell lines for functional odontoblastic features. The screening for odontoblast characteristic genes (detailed in Materials and methods) resulted in the selection of a single cell cloned, quickly proliferating (doubling time around 24 h) cell line. The immortalized odontoblast-lineage cells are shown in Fig. 1. OLCs are uniformly shaped elongated cells (80–100 lm), and they tend to form a swirling-like, wavy pattern near to confluence. Mature odontoblasts are described as columnar shaped, polarized cells with a characteristic long process towards the forming dentin in vivo. As illustrated in Figs. 1B and
Fig. 1. Morphology of mouse tooth germ derived odontoblast-lineage cells (OLC). (A) Phase contrast microscopic image of homologous OLC cells at confluence, 3 days in culture. (B) Higher magnification shows homologous, fibroblast-like shaped cells. Note the presence of some cells with long cellular processes (indicated by arrows). (C) Green fluorescent protein detection in OLCs by fluorescence microscopy. (D) Micrograph of a long cellular process (arrow) taken by confocal microscope.
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D, some OLCs exhibited long cellular processes, suggesting cell polarization, which is similar to the above-described odontoblast cell shape. Morphologically, OLCs resemble the primary cells from rat tooth germ isolated by Hao et al. [1]. However, they were different in shape from the polygonal, epitheloid MDPC-23 odontoblast-like cells. Odontoblast like gene expression analysis of the selected cell line To examine whether the isolated cloned cells belong to odontoblast-lineage and to clarify their gene expressing properties, we employed RT-PCR approach. We created a gene expression palette (as listed in Materials and method), to model a representative gene expression of in vivo odontoblast cells, since solely dentin-specific genes are not described in the literature at the moment. Primary cultured and cloned cells were subjected to RT-PCR analysis, which, from the results of OLC passage 10, is described in Fig. 2 (the findings for subsequent transfers were consistent with it). Our established cell line was positive for various odontoblast representative transcripts from the SIBLING protein family such as DSPP, DMP-1, MEPE, and osteopontin. The expression profile of these genes, shown in Figs. 2A
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and B, is closely similar to those observed in the total RNA extracted from mouse tooth germ ppd1, which served as a positive control in our experiments. Furthermore, transcripts of procollagen-1 and BMP-4 were also recognized in OLCs (Fig. 2B). In the present study, dentin ECM markers described by previous publications were investigated. We therefore established a gene expression profile for OLC, focused particularly on DSPP; (thought to be dentin specific protein until the idea’s recent denial [23]); the most significant non-collagenous dentin extracellular matrix (ECM) protein besides DMP-1, MEPE; a recently identified dentin/bone matrix protein [24] and osteopontin; a phosphoprotein expressed by osteoblasts and odontoblasts. According to this, OLCs demonstrated more complete dentin specific gene expression model compared to other known odontoblast-like cell lines [6–8]. Lhx6 and Lhx7 expression based on presumptive odontogenic mesenchyme origin Next, we evaluated Lhx6 and Lhx7 gene expressions of OLCs. As presented in Fig. 2C, positive signals were detected in OLCs as in tooth germ controls. We aimed to define the gene expression of Lhx6 and Lhx7 in OLCs on
Fig. 2. Gene expression analysis of OLCs by RT-PCR. (A) Detection of DSPP and DMP-1with GAPDH (housekeeping gene). Lane 1, ppd1 tooth germ (positive control); lane 2, mouse adult skin fibroblast (negative control); lane 3, OLC cultured in a-MEM. (B) ECM specific transcripts and GAPDH controls in OLC total RNA compared with tooth germ total RNA. (C) Lhx6 and Lhx7 gene expression pattern in OLC.
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the basis of the following. Grigoriou et al. [20] revealed restricted expression of both genes in neural-crest-derived mesenchyme in tooth germ from different dental development stages by in situ hybridization. They detected Lhx6 and Lhx7 transcripts in the odontogenic mesenchyme at the bell stage of developing teeth, which is equivalent with the developmental stage of tooth germs used in our study. According to Priam et al. [19] Lhx6 and Lhx7 mRNAs are limited to dental-pulp derived cells, and could not be detected in other mesodermal or stromal progenitor cells. Considering our positive gene expression data, we would like to suggest that our novel cell line is most likely arising from neural crest-derived odontogenic-potential cells. DSP and DMP-1 protein expression of OLC In order to investigate the production of dentin extracellular matrix proteins, we performed immunohistochemical analysis. Intracellular localization of DSP and DMP-1
proteins is demonstrated in Fig. 3A. Non-specific bindings were not found using controls without primary antibodies (data not shown). We detected immunofluorescence signals for both proteins in cell cytoplasm, particularly evident in the perinuclear region, and observed co-expression of DSP and DMP-1 in OLC. These findings were verified by Western blotting, hence we could detect representative bands for DSP (95 kDa in Fig. 3B) as well as DMP-1 (37 and 57 kDa proteolysis fragments in Fig. 3C). Together these results provide evidence that OLCs secrete DSP and DMP-1 proteins, which are known components of dentin ECM in vivo. As previous studies have already indicated [12,25] DMP1 expression has been detected in young odontoblast, in some dental papilla cells and weakly in mature odontoblasts, but not in preodontoblast, Bronckers et al. [26] have detailed the DSP expression in young and mature odontoblast and also in most dental papilla cells. Although our approach did not allow precise localization of our cells in the tooth germ, based
Fig. 3. Expressions of dentin specific proteins in OLC. (A) Immunostaining of OLCs with specific antibodies. Samples were collected after 1 week cultivation on collagen type-I coated coverslips and examined by confocal microscopy. (Nuclear staining is an artifact due to Triton X-100 treatment of cells.) (B) Western blotting was performed from protein extracts of OLC culture and mouse tooth germs (positive control).
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on the above-mentioned observations we suspect that their possible source might be either among young odontoblast cells or among those predetermined DSP positive immature dental papilla cells. The latter might refer to some potential precursors of odontoblast-lineage cells described by Ruch et al. [16] and Kikuchi et al. [17] in the marginal zone of murine dental papillae. ‘‘Osteogenic differentiation medium’’ induced calcification of OLC cultures in vitro For in vitro calcification study, OLCs were plated on collagen type-I-coated plates and supplemented with lowconcentration biomineralization factors (2 mM b-glycerophosphate, 50 lg/ml ascorbic acid) or high-concentration biomineralization factors (10 mM b-glycerophosphate, 50 lg/ml ascorbic acid) in a-MEM. In 3 days, the cells grew as monolayer, then created multiple layers. The first sign of calcification was found after 14 days of cultivation; then after 28 days we detected depositions of phosphate and calcium by alizarin red S, in Fig. 4A. Microscopic examination (Fig. 4B) revealed that, mineralization centers exhibited increased level of mineral deposition while the surrounding peripheral zone showed less advanced matrix mineralization. In this study, both low- and high-concentration medium initiated mineral deposition. However, cultured cells on high-concentration showed more prominent mineralization compared to those low-concentration
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cultures. These results suggest that b-glycerophosphate and ascorbic acid induce biomineralization of OLCs, and the degree of that is b-glycerophosphate concentration dependent. To address the question whether of Lhx6 and Lhx7 mRNA levels are affected by in vitro calcification, we examined their expression before and after mineralization. Lhx6 transcript level decreased to 0.7-fold (2 mM b-glycerophosphate) and to 0.3-fold (10 mM b-glycerophosphate) compared with controls (non-treated cells) determined by real-time PCR method. On the other side, Lhx7 expression showed only a slight drop in both groups, which did not exceed 0.7-fold alteration compared to controls. This study included the first attempt, to our knowledge, to follow Lhx6 and Lhx7 expression during biomineralization. As defined earlier, throughout mouse tooth development [20], high levels of Lhx6 and Lhx7 were detected until the bell stage, which was followed by down-regulated expression at ppd2. Assuming that odontoblast differentiation eventulizes during the late bell stage (around 6 h period in mice [27]) simultaneously with the onset of dentinogenesis, our observation in Lhx gene expression might reflect that critical developmental period in tooth morphogenesis. In summary, the present study reports on a novel odontoblast-lineage cell line established from mouse embryo tooth germs. Presumably, OLCs are neural-crest origin cells, residing in the dental papilla. OLCs were detected
Fig. 4. Induction of mineral deposition in OLC cultures. Calcification was detected by alizarin red S staining. (A) Effects of 2 mM (low concentration) or 10 mM (high concentration) b-glycerophosphate and 50 lg/ml ascorbic acid supplementation (28 days). Control cells show no spontaneous calcification. (B) Photomicrographs illustrating the progression of biomineralization: 1, initial phase at 15 days; 2, the effect of 2 mM b-glycerophosphate treatment at 28 days; 3, strong stains of mineralized matrix with 10 mM b-glycerophosphate treatment.
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to express dentin matrix proteins and were also positive for novel odontoblast phenotype markers such as Lhx6 and Lhx7. OLCs are rapidly proliferating cells with biomineralization capability in adequate conditions in vitro. Thus, we believe that this novel cell line produced on the basis of recent evidence and achievements in dentin biochemistry could serve beneficially for an odontogenesis model. Our findings provide spontaneously immortalized odontoblast-lineage cells with odontoblast phenotypic properties for future studies on dental development. Tissue recombination experiments might principally profit on the suitable tracing feasibility based on the green florescence protein (GFP) inclusion of this novel cell line. Acknowledgments This study was supported in part by a grant for the Research for the Future Program from the Japan Society for the Promotion of Science, by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology. References [1] J. Hao, K. Narayanan, A. Ramachandran, G. He, A. Almusthayt, C. Evans, A. George, Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue both in vitro and in vivo, J. Biol. Chem. 277 (2002) 19976–19981. [2] H.H. Ritchie, D.G. Ritchie, L.H. Wang, Six decades of dentinogenesis research, Eur. J. Oral Sci. 106 (Suppl. 1) (1998) 211–220. [3] M.Y. Kuo, W.H. Lan, S.K. Lin, K.S. Tsai, L.J. Hahn, Collagen gene expression in human dental pulp cell cultures, Arch. Oral Biol. 37 (1992) 945–952. [4] M.L. Couble, J.C. Farges, F. Bleicher, B. Perrat-Mabillon, M. Boudeulle, H. Magloire, Odontoblast differentiation of human dental pulp cells in explant cultures, Calcif. Tissue Int. 66 (2000) 129–138. [5] S.W. Whitson, D.B. Jenkins, D.E. Bowers Jr., J.F. Hatton, The isolation and primary culture of putative human root odontoblasts, Proc. Finn Dent. Soc. 88 (Suppl.1) (1992) 305–311. [6] S. Kasugai, M. Adachi, H. Ogura, Establishment and characterization of a clonal cell line (RPC-C2A) from dental pulp of the rat incisor, Arch. Oral Biol. 33 (1988) 887–891. [7] T. Kawase, M. Orikasa, A. Suzuki, A clonal prostaglandin-responsive cell line (RDP 4-1) derived from rat pulp, Bone Miner. 11 (1991) 163–175. [8] C.T. Hanks, Z.L. Sun, D.N. Fang, C.A. Edwards, J.C. Wataha, H.H. Ritchie, W.T. Butler, Cloned 3T6 cell line from CD-I mouse fetal molar dental papillae, Connect. Tissue Res. 37 (1998) 233– 249. [9] W. He, Z. Niu, S. Zhao, A. Smith, Smad protein mediated transforming growth factor beta1 induction of apoptosis in the MDPC-23 odontoblast-like cell line, Arch. Oral Biol. 50 (2005) 929–936. [10] C.A. de Souza Costa, J. Hebling, R.C. Randall, In vitro cytotoxicity of five glass-ionomer cements, Biomaterials 24 (2003) 3853– 3859.
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Phenotype properties of a novel spontaneously immortalized odontoblast-lineage cell line Szilvia Arany *, Akira Nakata, Takashi Kameda, Souchi Koyota, Yasuharu Ueno, Toshihiro Sugiyama Department of Biochemistry, Akita University School of Medicine, Hondo 1-1-1, Akita 010-8543, Japan Received 1 February 2006 Available online 14 February 2006
Abstract Here we report on the spontaneous immortalization upon serial passages of mouse fetal dental papilla cells, which present odontoblast phenotype features. The cells named odontoblast-lineage cell (OLC) produced dentin extracellular matrix proteins, such as DSP and DMP1, and maintained transcripts of various matrix components as osteopontin, BMP-4, procollagen-1, and MEPE. The addition of osteogenic differentiation medium with b-glycerophosphate and ascorbic acid was effective for inducing calcification and mineralization in vitro in cell cultures for up to 28 days. For the first time, we investigated the expression of Lhx6 and Lhx7 genes during induced biomineralization, since these new members of LIM homeodomain proteins have been recently proposed tracking odontoblastic phenotypes. Our results indicate that b-glycerophosphate treatment of OLC cultures decreases Lhx6 transcript levels in vitro. Our findings proved odontoblast phenotype-specificity, which demonstrates that this novel odontoblast-lineage cell line is a valuable tool for future experiments in odontology. 2006 Elsevier Inc. All rights reserved. Keywords: Odontoblast; Spontaneous immortalization; Dental papilla; Dentin extracellular matrix proteins; Calcification; Lhx6; Lhx7
To create models of dental development and to elucidate epithelial–mesenchymal interactions and pathways of tooth formation, continuous cell lines providing consistent and homogeneous research material are inevitably necessary [1]. These cell lines are considered to be useful tools for tissue engineering purposes, tissue recombinant studies, dental restorative, repairment experiments, as well as, research utilities for dental material testing. Therefore, several attempts focused on the isolation and the production of tooth cells applicable also for cytodifferentiation and biomineralization experiments. Among them, numerous studies shed some light on the dentinogenesis responsible odontoblasts, which are neural crest-derived mesenchymal cells from the first branchial arch. Their differentiation during tooth development and maturation is the result of the signaling cascade between the inner dental epithelium and *
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0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.02.020
the adjacent dental mesenchyme. However, to date, the complexity of the molecular mechanism underlying those interactions is not fully understood [2]. Putative odontoblast progenitor cells have been recognized [3,4] and predetermined odontoblastic population in dental pulp has been described [5] earlier. These systems were principally suitable for the analysis of odontoblast differentiation from tissue explants, although they did not yield continuous cell lines. According to the relevant literature, existing rat dental pulp cell lines; RPC-C2A [6] and RDP 4-1 [7] showed an incomplete odontoblast specific gene expression pattern. A spontaneously transformed mouse dental papilla cell line, called MDPC-23, developed by Hanks et al. [8], has already been chosen for various experiments [9,10] and proven its usefulness and applicability for dental research. It expresses common proteins of mineralizing dentin, as collagen, osteopontin, as well DSP, and inducible to form mineralized nodules in vitro. However, the authors were
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unable to detect transcripts for DMP-1 from MDPC-23 cells, which likely regulates ECM mineralization process in vivo and plays a significant role in terminal differentiation of odontoblasts [11]. Additionally, several authors reported on the advantages of using dental pulp stem cells (DPSC) in topics related to dentinogenesis [12,13]. Those cells obtained from dental pulp can be genetically transformed for odontoblast-like cell required experiments [14]. However, none of those approaches resulted in continuous cell lines from spontaneous immortalization, yet the potential side effects of applied viral oncogenes should be considered. Our laboratory reported on the successful establishment of an ameloblast-lineage cell line (ALC) [15], which represents the epithelial component of tooth germs. As a consecutive part of that study, the present research attempted to develop a continuous cell line from the mesenchymal component of tooth germ, which is devoted to odontoblast development. It has been proposed that a layer of odontoblast-lineage cells is located at the periphery of murine dental papillae [16,17], which encouraged us to employ mouse tooth germ explants as described in details in this paper. Here we present the isolation and the spontaneous immortalization of an odontoblast-lineage cell line, which has been probed to match functional criteria of odontoblasts. Hence, we searched transcripts for some of the small integrin binding ligand N-linked glycoprotein (SIBLING) protein genes, which are believed to play important role in osteogenesis and dentinogenesis [18]. Among the SIBLING proteins, we assayed our cells for DSPP, DMP-1, MEPE, and osteopontin transcripts. We included gene expression experiments on known markers for dentin extracellular matrix (ECM): procollagen-1 (PCOL-1) and BMP-4. Moreover, we tested our established cell line concerning a novel discovery in recognizing odontoblast phenotype. According to a recent advancement, transcripts of Lhx6 and Lhx7 were expressed in dental clones and postulated to be applicable markers of cell lines maintaining properties of odontoblastic phenotype [19]. Lhx6 and Lhx7 are members of a novel subfamily of LIM homeodomain proteins, which have already been detected in the presumptive odontogenic mesenchyme [20]. We performed an experiment to determine whether in vitro, induced calcification with ‘‘osteogenic differentiation medium’’ [21] alters the LIM gene expressions. Materials and methods Cell culture experiments. All experiments on animals were conducted with the approval of the Committee on Animal Experimentation of Akita University. Tooth germs were dissected out from 18.5-day-old C57BL/ 6–TgN (act-EGFP) OsbC14–YO1–FM131 mouse embryos under microscopic observation. Following aseptic isolation, washing with phosphatebuffered saline (PBS), and seeding on type-I collagen coated 60 mm culture dishes (Corning, USA), primary cell cultures were obtained from explants’ outgrowth after 4 weeks. After removing the tooth germs, fibroblastic-like outgrowths were cultured in minimum essential medium alpha modification (a-MEM) (Sigma, USA) supplemented with 15% (v/v) heat-inactivated
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fetal bovine serum (Sigma. USA), and fibroblast growth factor (FGF)-2 (2 ng/ml) on type-I collagen coated 6-well culture plates at 37C in a humified chamber of 5% (v/v) CO2 in air. The spread cells were transferred to new dishes at subconfluence, after 0.05% trypsin–0.02% EDTA digestion and maintained as described previously by Hanks et al. [8]. At passage 15, cells were inoculated at low density (10–15 cells per 60 mm dish) and single cell derived colonies were established for further cultivation. Those subcultures were studied routinely under an inverted microscope. Twenty colonies were picked and investigated for odontoblast phenotype (detailed later in this section). Subsequently, the selected odontoblast-lineage cells (OLCs) were transferred over 60 times to present. RNA preparation, reverse transcriptase-PCR, and quantitative RTPCR. Total RNA was extracted from single cell cloned colonies, likewise OLC cultures after every tenth passage using ISOGEN reagent (Nippon Gene, Japan) according to the manufacturer’s protocol. Concentrations of total RNA were determined by the absorption ratio (OD260/OD280 nm) with UV spectrophotometry (Beckman, Germany). Then the template RNA were processed to evaluate odontoblastic gene expression pattern using the Superscript One-Step RT-PCR with Platinum Taq system (Invitrogen, USA). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. Reaction mixtures that were set up included specific mouse primers sets (10 pM/ll) for dentin sialophosphoprotein (DSP) (5 0 -ACAGCAAATGGCATCAGGAG-3 0 and 5 0 -CT CGTAACTGTACCCGTACT-3 0 ), dentin matrix protein (DMP)-1 (5 0 -GAAAGCTCTGAAGAGAGGAC-3 0 , 5 0 -GTCATCTGAATCCG 0 0 TAGTGC-3 ), procollagen-1 (5 -CTGCCGGAGAAGAAGGAAAA-3 0 , 5 0 -CCATCTTTACCAGGAGAACC-3 0 ), osteopontin 5 0 -TGGACGAC GATGARGACGAT-3 0 , 5 0 -GACTATCGATCACATCCGAC-3 0 ), bone morphogenetic protein (BMP)-4 (5 0 -AGGCGACACTTCTACAGATG3 0 , 5 0 -ATGGCCAAAAGTGACCAGGA-3 0 ), matrix extracellular phosphoglycoprotein (MEPE) (5 0 -GCTCCAGCAAAGCTGAAG-3 0 , 5 0 -AC TAAGACTGTGAGAGGGCA-3 0 ), Lhx6 (5 0 -AATCTATGCCAGTGA CTGGG-3 0 , 5 0 -GTGTCGTAATGGATGCGACA-3 0 ), and Lhx7 0 (5 -TACTTCAGACGGTATGGGAC-3 0 , 5 0 -TCCCATTACCGTTCTCC ACT-3 0 ). We designed the primers using information from GenBank database (http://www.ncbi.nlm.nih.gov/Genbank). The reverse transcriptase PCR conditions were set up as follows: 35 cycles of 60 s denaturation at 94 C, 60 s annealing at 56–58 C, and 90 s extension at 70–72 C. Appropriate negative controls (mouse fibroblast cells for DSP and DMP1) and positive controls (total RNA isolated from mouse tooth germs from post-partum day, ppd1) were included in all examinations. The PCR products were loaded on 1.5% agarose gel for electrophoresis and visualized by ethidium bromide staining under ultraviolet light exposure. The quantification of Lhx6 and Lhx7 transcripts was performed by real-time PCR method using the SuperScript Platinum III Two-Step qRT-PCR Kit with SYBR Green (Invitrogen, USA). For LightCycler experiments (Roche Molecular Biochemicals, Switzerland) 2 ll cDNA template (reverse transcribed total RNA from 2 or 10 mM b-glycerophosphate administered cultures) was added to 18 ll master mix containing the specific primer pairs. The following LightCycler programming was used: initial denaturation (95 C for 2 min), 45 cycles of amplification with a single fluorescent measurement (denaturing at 94 C for 5 s, annealing at 58 C for 10 s, and elongation at 72 C for 10 s), and melting curve analysis with continuous fluorescent measurement. Finally, standard curves from control dilution series were created with LightCycler Software, Version 3.5. The relative quantity of target genes was calculated by the normalization method versus 18S reference gene. Western blotting and immunofluorescence microscopy. Cultured cells at confluence were lysed in PhosphoSafe Extraction Buffer (Novagen, USA) and centrifuged for 5 min at 16,000g at 4 C. The lysate concentration was assayed using the BCA method, and then the protein samples were subjected to SDS–PAGE gel electrophoresis on 10% (w/v) acrylamide gel. Separated proteins were electrophoretically transferred onto PVDF membrane (Immobilion-P, Millipore, USA) with a semi-dry system. After rinsing and blocking (5% dried skim milk) the blots DSP and DMP-1 were detected by commercially available antibodies: by DSP goat polyclonal antibody (Santa Cruz Biotechnology, USA) and by DMP-1 rabbit
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monoclonal antibody (Takara, Japan), respectively. The proteins were visualized by enhanced chemiluminescence (ECL) (Amersham, Sweden) according to the manufacturer’s instructions. For immunohistochemical analysis, cells were cultivated on collagen type-I coated glass coverslips inserted in 24-well plates, and, after reaching confluence, they were fixed in 4% paraformaldehyde. Following permeabilization with 0.1% Triton X-100 in PBS and subsequent blocking, the samples were incubated at 4 C overnight with primary antibodies against DSP and DMP1, diluted 1:300 in blocking solution. Then samples were washed in PBST and incubated with the secondary antibodies; Alexa Fluor 633-conjugated anti-goat antibody (Molecular Probes, USA) and Alexa Fluor 546 anti-rabbit antibody, respectively, diluted 1:500 in PBST. The sections were washed with PBST and mounted onto microscopic slides. Images were obtained and analyzed using the LSM-510 confocal imaging system (Zeiss, Germany). Induction of in vitro mineralization and alizarin red staining. To induce calcification and nodule formation, cells were seeded at a density of 3 · 104 and cultured in conditioned a-MEM medium with 2 mM (low concentration) or 10 mM (high concentration) b-glycerophosphate plus 50 lg/ml ascorbic acid (Sigma, USA) for 4 weeks. The control group was cultured in a-MEM containing 15% FBS. The medium was refreshed every 3 days. On day 28, the cultures were fixed in 4% paraformaldehyde for 10 min and then stained with 1% alizarin red S (Sigma, USA).
Results and discussion Establishment and morphology of cultured mouse tooth germ derived cells To establish immortalized dental cell line with odontoblastic phenotype, tooth germs removed from E18.5 mouse mandibles were placed in culture medium. On the first day,
tooth germs attached to the collagen coated dish, and during the following days outgrowth of dental papilla cells could be observed in most of the dishes. The proliferating cells emerged in a thickening area surrounding the central germ, which was discarded after 1 week. We observed mainly two types of outgrowth, distinguished by cell shape. The spreading cells showed either a polygonal-shaped or a fibroblast-shaped appearance. For further transfer, we selected explants that contained cells exhibiting fibroblastic-like morphology with vigorous outgrowth during 4 weeks. In order to design this experiment, we also referred to a previously described 3T3 regimen [22] of routine subculture. We seeded cells at low density to perform dilution cloning at passage 15. Twenty of the isolated cell populations were collected with cloning rings and allowed to grow to subconfluence. From this point, we continued the regular schedule (serial passage with 3 days interval) on cell transfer and investigated the cell lines for functional odontoblastic features. The screening for odontoblast characteristic genes (detailed in Materials and methods) resulted in the selection of a single cell cloned, quickly proliferating (doubling time around 24 h) cell line. The immortalized odontoblast-lineage cells are shown in Fig. 1. OLCs are uniformly shaped elongated cells (80–100 lm), and they tend to form a swirling-like, wavy pattern near to confluence. Mature odontoblasts are described as columnar shaped, polarized cells with a characteristic long process towards the forming dentin in vivo. As illustrated in Figs. 1B and
Fig. 1. Morphology of mouse tooth germ derived odontoblast-lineage cells (OLC). (A) Phase contrast microscopic image of homologous OLC cells at confluence, 3 days in culture. (B) Higher magnification shows homologous, fibroblast-like shaped cells. Note the presence of some cells with long cellular processes (indicated by arrows). (C) Green fluorescent protein detection in OLCs by fluorescence microscopy. (D) Micrograph of a long cellular process (arrow) taken by confocal microscope.
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D, some OLCs exhibited long cellular processes, suggesting cell polarization, which is similar to the above-described odontoblast cell shape. Morphologically, OLCs resemble the primary cells from rat tooth germ isolated by Hao et al. [1]. However, they were different in shape from the polygonal, epitheloid MDPC-23 odontoblast-like cells. Odontoblast like gene expression analysis of the selected cell line To examine whether the isolated cloned cells belong to odontoblast-lineage and to clarify their gene expressing properties, we employed RT-PCR approach. We created a gene expression palette (as listed in Materials and method), to model a representative gene expression of in vivo odontoblast cells, since solely dentin-specific genes are not described in the literature at the moment. Primary cultured and cloned cells were subjected to RT-PCR analysis, which, from the results of OLC passage 10, is described in Fig. 2 (the findings for subsequent transfers were consistent with it). Our established cell line was positive for various odontoblast representative transcripts from the SIBLING protein family such as DSPP, DMP-1, MEPE, and osteopontin. The expression profile of these genes, shown in Figs. 2A
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and B, is closely similar to those observed in the total RNA extracted from mouse tooth germ ppd1, which served as a positive control in our experiments. Furthermore, transcripts of procollagen-1 and BMP-4 were also recognized in OLCs (Fig. 2B). In the present study, dentin ECM markers described by previous publications were investigated. We therefore established a gene expression profile for OLC, focused particularly on DSPP; (thought to be dentin specific protein until the idea’s recent denial [23]); the most significant non-collagenous dentin extracellular matrix (ECM) protein besides DMP-1, MEPE; a recently identified dentin/bone matrix protein [24] and osteopontin; a phosphoprotein expressed by osteoblasts and odontoblasts. According to this, OLCs demonstrated more complete dentin specific gene expression model compared to other known odontoblast-like cell lines [6–8]. Lhx6 and Lhx7 expression based on presumptive odontogenic mesenchyme origin Next, we evaluated Lhx6 and Lhx7 gene expressions of OLCs. As presented in Fig. 2C, positive signals were detected in OLCs as in tooth germ controls. We aimed to define the gene expression of Lhx6 and Lhx7 in OLCs on
Fig. 2. Gene expression analysis of OLCs by RT-PCR. (A) Detection of DSPP and DMP-1with GAPDH (housekeeping gene). Lane 1, ppd1 tooth germ (positive control); lane 2, mouse adult skin fibroblast (negative control); lane 3, OLC cultured in a-MEM. (B) ECM specific transcripts and GAPDH controls in OLC total RNA compared with tooth germ total RNA. (C) Lhx6 and Lhx7 gene expression pattern in OLC.
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the basis of the following. Grigoriou et al. [20] revealed restricted expression of both genes in neural-crest-derived mesenchyme in tooth germ from different dental development stages by in situ hybridization. They detected Lhx6 and Lhx7 transcripts in the odontogenic mesenchyme at the bell stage of developing teeth, which is equivalent with the developmental stage of tooth germs used in our study. According to Priam et al. [19] Lhx6 and Lhx7 mRNAs are limited to dental-pulp derived cells, and could not be detected in other mesodermal or stromal progenitor cells. Considering our positive gene expression data, we would like to suggest that our novel cell line is most likely arising from neural crest-derived odontogenic-potential cells. DSP and DMP-1 protein expression of OLC In order to investigate the production of dentin extracellular matrix proteins, we performed immunohistochemical analysis. Intracellular localization of DSP and DMP-1
proteins is demonstrated in Fig. 3A. Non-specific bindings were not found using controls without primary antibodies (data not shown). We detected immunofluorescence signals for both proteins in cell cytoplasm, particularly evident in the perinuclear region, and observed co-expression of DSP and DMP-1 in OLC. These findings were verified by Western blotting, hence we could detect representative bands for DSP (95 kDa in Fig. 3B) as well as DMP-1 (37 and 57 kDa proteolysis fragments in Fig. 3C). Together these results provide evidence that OLCs secrete DSP and DMP-1 proteins, which are known components of dentin ECM in vivo. As previous studies have already indicated [12,25] DMP1 expression has been detected in young odontoblast, in some dental papilla cells and weakly in mature odontoblasts, but not in preodontoblast, Bronckers et al. [26] have detailed the DSP expression in young and mature odontoblast and also in most dental papilla cells. Although our approach did not allow precise localization of our cells in the tooth germ, based
Fig. 3. Expressions of dentin specific proteins in OLC. (A) Immunostaining of OLCs with specific antibodies. Samples were collected after 1 week cultivation on collagen type-I coated coverslips and examined by confocal microscopy. (Nuclear staining is an artifact due to Triton X-100 treatment of cells.) (B) Western blotting was performed from protein extracts of OLC culture and mouse tooth germs (positive control).
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on the above-mentioned observations we suspect that their possible source might be either among young odontoblast cells or among those predetermined DSP positive immature dental papilla cells. The latter might refer to some potential precursors of odontoblast-lineage cells described by Ruch et al. [16] and Kikuchi et al. [17] in the marginal zone of murine dental papillae. ‘‘Osteogenic differentiation medium’’ induced calcification of OLC cultures in vitro For in vitro calcification study, OLCs were plated on collagen type-I-coated plates and supplemented with lowconcentration biomineralization factors (2 mM b-glycerophosphate, 50 lg/ml ascorbic acid) or high-concentration biomineralization factors (10 mM b-glycerophosphate, 50 lg/ml ascorbic acid) in a-MEM. In 3 days, the cells grew as monolayer, then created multiple layers. The first sign of calcification was found after 14 days of cultivation; then after 28 days we detected depositions of phosphate and calcium by alizarin red S, in Fig. 4A. Microscopic examination (Fig. 4B) revealed that, mineralization centers exhibited increased level of mineral deposition while the surrounding peripheral zone showed less advanced matrix mineralization. In this study, both low- and high-concentration medium initiated mineral deposition. However, cultured cells on high-concentration showed more prominent mineralization compared to those low-concentration
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cultures. These results suggest that b-glycerophosphate and ascorbic acid induce biomineralization of OLCs, and the degree of that is b-glycerophosphate concentration dependent. To address the question whether of Lhx6 and Lhx7 mRNA levels are affected by in vitro calcification, we examined their expression before and after mineralization. Lhx6 transcript level decreased to 0.7-fold (2 mM b-glycerophosphate) and to 0.3-fold (10 mM b-glycerophosphate) compared with controls (non-treated cells) determined by real-time PCR method. On the other side, Lhx7 expression showed only a slight drop in both groups, which did not exceed 0.7-fold alteration compared to controls. This study included the first attempt, to our knowledge, to follow Lhx6 and Lhx7 expression during biomineralization. As defined earlier, throughout mouse tooth development [20], high levels of Lhx6 and Lhx7 were detected until the bell stage, which was followed by down-regulated expression at ppd2. Assuming that odontoblast differentiation eventulizes during the late bell stage (around 6 h period in mice [27]) simultaneously with the onset of dentinogenesis, our observation in Lhx gene expression might reflect that critical developmental period in tooth morphogenesis. In summary, the present study reports on a novel odontoblast-lineage cell line established from mouse embryo tooth germs. Presumably, OLCs are neural-crest origin cells, residing in the dental papilla. OLCs were detected
Fig. 4. Induction of mineral deposition in OLC cultures. Calcification was detected by alizarin red S staining. (A) Effects of 2 mM (low concentration) or 10 mM (high concentration) b-glycerophosphate and 50 lg/ml ascorbic acid supplementation (28 days). Control cells show no spontaneous calcification. (B) Photomicrographs illustrating the progression of biomineralization: 1, initial phase at 15 days; 2, the effect of 2 mM b-glycerophosphate treatment at 28 days; 3, strong stains of mineralized matrix with 10 mM b-glycerophosphate treatment.
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to express dentin matrix proteins and were also positive for novel odontoblast phenotype markers such as Lhx6 and Lhx7. OLCs are rapidly proliferating cells with biomineralization capability in adequate conditions in vitro. Thus, we believe that this novel cell line produced on the basis of recent evidence and achievements in dentin biochemistry could serve beneficially for an odontogenesis model. Our findings provide spontaneously immortalized odontoblast-lineage cells with odontoblast phenotypic properties for future studies on dental development. Tissue recombination experiments might principally profit on the suitable tracing feasibility based on the green florescence protein (GFP) inclusion of this novel cell line. Acknowledgments This study was supported in part by a grant for the Research for the Future Program from the Japan Society for the Promotion of Science, by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology. References [1] J. Hao, K. Narayanan, A. Ramachandran, G. He, A. Almusthayt, C. Evans, A. George, Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue both in vitro and in vivo, J. Biol. Chem. 277 (2002) 19976–19981. [2] H.H. Ritchie, D.G. Ritchie, L.H. Wang, Six decades of dentinogenesis research, Eur. J. Oral Sci. 106 (Suppl. 1) (1998) 211–220. [3] M.Y. Kuo, W.H. Lan, S.K. Lin, K.S. Tsai, L.J. Hahn, Collagen gene expression in human dental pulp cell cultures, Arch. Oral Biol. 37 (1992) 945–952. [4] M.L. Couble, J.C. Farges, F. Bleicher, B. Perrat-Mabillon, M. Boudeulle, H. Magloire, Odontoblast differentiation of human dental pulp cells in explant cultures, Calcif. Tissue Int. 66 (2000) 129–138. [5] S.W. Whitson, D.B. Jenkins, D.E. Bowers Jr., J.F. Hatton, The isolation and primary culture of putative human root odontoblasts, Proc. Finn Dent. Soc. 88 (Suppl.1) (1992) 305–311. [6] S. Kasugai, M. Adachi, H. Ogura, Establishment and characterization of a clonal cell line (RPC-C2A) from dental pulp of the rat incisor, Arch. Oral Biol. 33 (1988) 887–891. [7] T. Kawase, M. Orikasa, A. Suzuki, A clonal prostaglandin-responsive cell line (RDP 4-1) derived from rat pulp, Bone Miner. 11 (1991) 163–175. [8] C.T. Hanks, Z.L. Sun, D.N. Fang, C.A. Edwards, J.C. Wataha, H.H. Ritchie, W.T. Butler, Cloned 3T6 cell line from CD-I mouse fetal molar dental papillae, Connect. Tissue Res. 37 (1998) 233– 249. [9] W. He, Z. Niu, S. Zhao, A. Smith, Smad protein mediated transforming growth factor beta1 induction of apoptosis in the MDPC-23 odontoblast-like cell line, Arch. Oral Biol. 50 (2005) 929–936. [10] C.A. de Souza Costa, J. Hebling, R.C. Randall, In vitro cytotoxicity of five glass-ionomer cements, Biomaterials 24 (2003) 3853– 3859.
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