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Keywords:

  • ectopic mineralization;
  • pulp cells;
  • TGF-β3

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Several members of the transforming growth factor (TGF)-β superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-β1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-β3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-β3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-β3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-β3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-β3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-β3 soaked beads in vivo. These results indicate for the first time that TGF-β3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Developing tooth germ is a useful specimen for the elucidation of genetic pathways in dentinogenesis. During tooth formation, interactions between dental epithelial and mesenchymal cells promote tooth morphogenesis by stimulating a subpopulation of dental papilla cells to differentiate into odontoblasts. Further, pulp cells, particularly odontoblasts, have been found to be important in the dentin repair response following dental caries, tissue injury and trauma (Smith 2002), and are thought to arise from the proliferation and differentiation of a precursor population residing somewhere within the pulp tissue (Gronthos et al. 2000). However, the origin, nature and molecular mechanisms involved with the recruitment of these cells have not been well characterized.

Transforming growth factor-β (TGF-β) is a multifunctional regulator of a variety of cellular functions, including proliferation, lineage determination, differentiation, motility, adhesion, apoptosis and extracellular matrix formation, and plays an important role in regulating tissue repair and regeneration (Roberts & Sporn 1993; Massague 1998). TGF-β molecules have also been implicated as key participants in odontoblast differentiation and dentin mineralization, as differentiated odontoblasts secrete and deposit TGF-β into the dentin matrix and can respond to it, thus enabling a possible autocrine action. TGF-β1 is normally expressed in both healthy and carious molar teeth in humans (Sloan et al. 2000). In rabbit dentin, the expression of TGF-β1 is greater than that of TGF-β3. TGF-β2 has only been detected in collagenase-released fractions and not in ethylenediamine tetraacetic acid (EDTA)-soluble fractions, whereas the expression of TGF-β1, but not TGF-β2 and TGF-β3, has been detected in human dentin matrix (Cassidy et al. 1997). In a previous study, when beads soaked with TGF-β1, β2 or β3 were placed on an area of odontoblasts in pulp from molars, predentin secretion and an increasing density of subodontoblasts cells occurred through the actions of TGF-β1 and TGF-β3, but not, however, through TGF-β2 (Sloan & Smith 1999).

Most studies of TGF-β and odontoblast differentiation have focused on the function of TGF-β1 because of its higher expression as compared with TGF-β2 and TGF-β3. TGF-β is known to stimulate the synthesis of extracellular matrices and initiate odontoblast differentiation in vitro and in vivo (Sloan et al. 2000). Further, bone and teeth contain type I collagen (COL I), as well as non-collagenous proteins such as osteocalcin (OCN) and dentin sialoprotein (DSP), and the expression of those matrices is regulated by TGF-β1. These sialic acid-rich proteins are also associated with the process of mineralization. Mineralized tissues are formed by matrix-mediated mineralization mechanisms, in which COL I forms the structural template for the epitaxial nucleation of hydroxyapatite (Beniash et al. 2000). Characterization of the molecular differences of matrix proteins and their expression patterns, along with the molecular control of the biosynthetic events that accompany these processes, are necessary to understand the mechanisms involved in mineralization. However, the molecular mechanisms of TGF-β, especially TGF-β3, involved with odontoblast differentiation and mineralization in the pulpal region are not clearly understood though it is known that the stem cells of odontoblasts that form dentin exist in dental pulp (Tziafas et al. 2000).

In the present study, we evaluated the pharmacological ability of TGF-β3 to induce ectopic mineralization and odontoblast differentiation in dental samples during tooth germ development. Beads soaked with TGF-β3 were implanted into fetal mouse tooth germs and cultured in serum-free conditions, or TGF-β3 was added exogenously to the culture medium. We found that TGF-β3 induced mineralization in the pulpal tissue. Further, an enhancement of the expression of COL I and OCN was observed. In addition, in a primary culture of dental mesenchymal cells taken from fetal dental papilla, which are known to contain dental pulp stem cells, the expression levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) were increased after TGF-β3 administration, whereas those results were not seen in calvaria osteoblasts. These findings indicate that TGF-β3 is important for the differentiation of odontoblasts and mineralization in dental pulp. The present data may also provide benefits for biological therapy for pulpal exposure that results from dental caries or tooth fracture.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mouse tooth germ organ culture

Lower first molar tooth germs were harvested from CBA mouse fetus (Nihon Clea, Tokyo, Japan) on embryonic day (E)17 and placed into a Rowell-type organ culture system using serum-free, chemically defined BGJb medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 100 µg/mL of ascorbic acid, 100 U each of penicillin and streptomycin and 1 mM beta-glycerophosphate, and then allowed to develop at 37°C under an atmosphere with 5% CO2 overnight, as described previously (Slavkin et al. 2000). Animal handling conformed to the guidelines of the Council on Animal Care, Kyushu University. Affi-Gel Blue Gel agarose beads (Bio-Rad Laboratories, Hercules, CA, USA) at 100 meshes (50 µm in diameter) were soaked overnight in 10 ng/µL or 100 ng/µL of TGF-β3 (Sigma-Aldrich, Steinheim, Germany) or in phosphate-buffered saline (PBS) as a control. The soaked beads were positioned as described previously (Nonaka et al. 1999) and shown in Fig. 2(a). Explants were cultured for 4 days, with the medium changed every 48 h. A total of 32 tooth germs were cultured and subdivided into four groups of eight tooth germs each for use in the experiments. After 4 days the tissues were harvested and processed for further histological examination.

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Figure 2. Induction of TGF-β3 in pulp cells by TGF-β3. TGF-β3 or PBS soaked beads (arrow head) were implanted in the tooth germ (a). Implanted bead (arrow head), dental pulp, predentin and stellate reticulum were identified by histological examination (b). No expression of TGF-β3 was detected in dental pulp (c). There was no expression of TGF-β3 in the pulpal area when the tooth germs were cultured in the presence of PBS-soaked beads (d), whereas pulp cells in the areas of the TGF-β3-soaked bead were stained positively with the TGF-β3 antibody (e,f). Bars: (a) 250 µm; (b) 50 µm; (c) 5 µm; (d–f) 25 µm.

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Pulpal mesenchymal cell culture

Tooth germs of the first lower molars of E18.5 fetal mice were treated with 0.1% collagenase, 0.05% trypsin, and 0.5 mM EDTA in 0.1 M PBS for 10 min at 37°C using pipetting. Mouse osteoblasts were obtained from newborn calvaria (Yuasa et al. 2004). Dental papilla cells and calvaria osteoblasts were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) with 2% fetal calf serum with or without 100 ng/mL of TGF-β3, for 48 h, as reported previously (Yuasa et al. 2004). Exogenous TGF-β3 was added to serum-free culture medium that contained Dulbecco's modified Eagle Medium (Invitrogen) supplemented with 5 µg/mL of insulin, 5 µg/mL of transferrin, 50 µg/mL of ascorbic acid, and 1 mg/mL of hydrocortisone. The culture medium was changed every other day and TGF-β3 was added when the medium was changed, and the cells were allowed to develop for 7 days.

Histological examination

The cultured tooth germs were kept in 4% paraformaldehyde with 0.01 M PBS for 24 h at 4°C. Fixed specimens were decalcified using 10% EDTA with 0.1% Tris-HCl/0.01 M PBS for 4 days. After dehydration through a graded series of ethanol and xylene solutions, the specimens were embedded in paraffin, and serial sections 4 µm thick were cut and collected for histological examination. After being deparaffinized, the slides were stained with hematoxylin-eosin and the stained sections were mounted using malinol (Muto Pure Chemistry, Tokyo, Japan). To analyze calcium deposition, non-decalcified specimens from organ cultured tooth germs were used. Briefly, the sections were stained with 0.5% Alizarin Red S solution (Sigma, St. Louis, MO, USA) for 15 min at room temperature. The stained sections were then washed threefold with distilled water, dehydrated by ethanol, and finally mounted using malinol.

Immunohistochemistry

Antibodies against TGF-β3 (1:150; Santa Cruz Biotechnology, Santa Cruz, CA, USA), OCN (1:400; L.S.L. Tokyo, Japan), and COL I (1:1000; Cosmo Bio, Tokyo, Japan) were used. Immunohistochemistry was performed as described previously (Kohama et al. 2002). The negative controls included sections incubated with normal rabbit serum or normal goat serum, instead of the primary antibody. The intensity of the immunoreaction for OCN and COL I was measured as previously described (Nonaka et al. 1999). Briefly, the photographs were scanned and the outlines of the stained area traced, then the relative area was determined using NIH Image version 1.6.1 (NIH, Bethesda, MD, USA). Five serial sections were obtained from each tooth germ organ culture, with five organ cultures used in each experiment.

Alizarin Red S staining

Alizarin Red S staining for mineralized nodules was performed as follows. Cultured cells were fixed with 99% ethyl alcohol (EtOH) for 5 min on ice. After washing threefold with de-ionized water (DW), the fixed cells were incubated with 1% Alizarin Red S (Sigma-Aldrich) in DW (adjusted to pH 4.1–4.3) for 20 min at room temperature and washed twice with DW and PBS. Stained cultures were phosphated followed by a quantitative distaining procedure using 10% cetylpyridinium chloride (CPC) in 10 mm sodium phosphate, pH 7.0, for 15 min at room temperature. Aliquots of these Alizarin Red S extracts were diluted 10-fold in 10% CPC after which the concentration of Alizarin Red S determined by absorbance measurement at 562 nm on a multiplate reader using a standard curve in the same solution. Results are reported as optical density (OD).

In situ hybridization

In situ hybridization was performed using a mouse digoxygenin (DIG)-labeled DSP RNA probe as previously described (Nakashima et al. 2002) with frozen sections. For the mouse probe, a 1080 bp clone (9586–10665) was obtained by polymerase chain reaction (PCR) from mouse genomic DNA (Clontech, Palo Alto, CA, USA) using the following primers, DSP, 5′-CGCGAATTCGACAGGAGAGATGTGCACACT-3′, and 5′-TACGGATCCAGGAGGTGAGCACCTGAGAA-3′, then subcloned into pBlueScript II SK (–), linearized with HindIII, and transcribed with T3 polymerase for the antisense probe.

Reverse transcription-polymerase chain reaction

The tooth germs, dental mesenchymal cells, and calvaria osteoblasts were cultured overnight in a serum-free culture system to prevent confounding variations induced by serum factors. Predetermined concentrations of TGF-β3 were exogenously added to these cultures. After 48 h of cultivation with exogenous TGF-β3, the cells were subjected to an additional RNA extraction, as previously described (Nonaka et al. 1999). Total RNA was isolated using TRIzol reagent, according to the manufacturer's instructions (Invitrogen). First strand cDNA was synthesized at 42°C for 90 min using oligodeoxythymidine 14 primers. Real time PCR amplification was performed using primers for COL I (5′-CCCAGAGTGGAACAGCGATTAC-3′ and 5′-TGTCTTGCCCCATTCATTTGTC-3′), dentin DSPP (5′-CTCAGAGAGAATCTGGGTGTACCACC-3′ and 5′-CACAGTGGTACATGGAGAGCTC-3′), DMP1 (5′-GCTTCAGGCTCAGTCTTGCT-3′ and 5′-TGTAACCCTCCAACTCCAGG-3′), osteonectin (ONC; 5′-GTCTCACTGGCTGTGTTGGA-3′ and 5′-AAGACTTGCCATGTGGGTTC-3′), osteopontin (OPN; 5′-CGATGATGATGACGATGGAG-3′ and 5′-GAGGTCCTCATCTGTGGCAT-3′), OCN (5′-CCTCTTGAAAGAGTGGGCTG-3′ and 5′-CCTCGGGAGACAAACAACAT-3′), and glyceraldehyde 3 phosphate dehydrogenase (G3PDH; 5′-CCATCACCATCTTCCAGGAG-3′ and 5′-GCATGGACTGTGGTCATGAG-3′), with SYBR Green PCR Master Mix and a TaqMan 7700 Sequencer Detection (Applied Biosystems, Foster City, CA, USA). PCR was performed for 40 cycles at 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min, as reported previously (Fukumoto et al. 2003). Concomitant mouse β-actin PCR (Genesetoligos, Kyoto, Japan) assays with the reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) primer sets described above were also performed for each RT reaction as normalization controls.

Statistical analysis

An unpaired two-tailed Student's t-test was performed on immunohistochemical intensity, RT-PCR expression level, and the OD of nodule formation results. Results of P < 0.05 were considered to be statistically significant in this study.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Induction of TGF-β3 in pulp cells by TGF-β3

Transforming growth factor-β3 mRNA was detected in rat incisor pulp cells, which are known to contain odontoblasts, by Northern blot analysis in our previous study (Nakashima et al. 1998). We also performed TGF-β3 immunohistochemistry using cultured mouse tooth germs. A positive reaction for TGF-β3 was detected in odontoblasts and ameloblasts, but not in dentin or enamel (data not shown). In the present study, no expression of TGF-β3 was detected in dental pulp (Fig. 2c). To determine the effect of TGF-β3 toward dental pulp cells, beads soaked in TGF-β3 or PBS soaked beads were placed on dental pulp regions, as shown in Figure 2(b). The experimental design for this study is summarized in Figure 1. There was no expression of TGF-β3 in the pulpal area when the tooth germs were cultured in the presence of PBS-soaked beads (Fig. 2d), whereas pulp cells in the areas of the TGF-β3-soaked bead were stained positively with the TGF-β3 antibody (Fig. 2e,f). TGF-β3 was found localized mostly in the cytoplasm. Further, we confirmed the inductive expression of TGF-β3 by RT-PCR. TGF-β3 mRNA expression was detected in the tooth germ cultured with TGF-β3-soaked beads, however, not in the intact or PBS-soaked controls (data not shown). These results suggest that TGF-β3 was produced by the pulp cells, and not released from the beads.

image

Figure 1. Schematic representation of the experimental design for Materials and Methods. The tooth germs were harvested for organ culture and cell culture and allowed to develop overnight. Beads were soaked overnight in 10 ng/µL, 100 ng/µL transforming growth factor (TGF)-β3 or phosphate-buffered saline (PBS), and were positioned in the pulpal area. The same amounts of tooth germ without bead implantation were cultured as intact controls. Explants were allowed to develop for 4 days for further histological examination, Alizarin Red S assay, immunohistochemistry and in situ hybridization. Moreover, PBS or two concentrations of TGF-β3 were exogenously added to organ culture explants. After 48 h of cultivation, tooth germs were subjected to a further RNA extraction to detect osteocalcin (OCN), type I collagen (COL I), and dentin sialoprotein (DSP). Furthermore, PBS or two concentrations of TGF-β3 were exogenously added to cell culture explants. After 7 days of cultivation, the cultured cells were incubated with 1% Alizarin Red S to detect nodule formation. Dental papilla cells and calvaria osteoblasts were cultured with or without 100 ng/mL TGF-β3 for 48 h. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers for dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), osteonectin (ONC), osteopontin (OPN), OCN and G3PDH.

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TGF-β3 induced mineralized tissue in pulp

The TGF-β family induces the formation of mineralized tissue during differentiation of osteoblasts and chondrocyte. To analyze the effect of TGF-β3 on mineralization in pulp tissue, we performed a tooth germ organ culture with beads soaked in TGF-β3 and histological analysis with hematoxylin and eosin (H&E) and Alizarin Red S staining. Eosin positive staining was observed around the TGF-β3-soaked beads (Fig. 3A,b), however, not around those soaked in PBS (Fig. 3A,a). These results indicate that the pulp cells may have secreted extracellular matrices around the TGF-β3-soaked beads.

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Figure 3. TGF-β3 induced mineralized tissue in pulp. (A) hematoxylin and eosin (H&E) staining results show that eosin positive staining was observed around the TGF-β3 soaked beads (b), but not in PBS (a). (B) Alizarin Red S staining was not observed around the PBS-soaked beads (a), whereas it was seen in the areas surrounding the beads soaked in TGF-β3 (b). The induction rate of ectopic mineralization was 0% (0/8) in both the intact controls and PBS-soaked beads, 25% (2/8) in 10 ng/µL of TGF-β3 and 87.5% (7/8) in those soaked with 100 ng/µL of TGF-β3 (C). Bars, 25 µm.

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Alizarin Red S staining was performed to identify mineralization that occurred in the dental pulp during culture. When PBS-soaked beads were implanted into the pulp tissue, Alizarin Red S staining was not observed around the beads (Fig. 3B,a), whereas it was seen in the areas surrounding the beads soaked in TGF-β3 (Fig. 3B,b), indicating that mineralization had occurred. The induction rate of ectopic mineralization was 0% (0/8) in both the intact controls and PBS-soaked beads, 25% (2/8) in 10 ng/µL of TGF-β3 and 87.5% (7/8) in those soaked with 100 ng/µL of TGF-β3 (Fig. 3C).

TGF-β3 induced the expression of OCN and type I collagen

Type I collagen and OCN are extracellular matrices known to be expressed in fully differentiated odontoblasts (Mina & Braut 2004), as well as in osteoblasts and osteocytes in alveolar and calvaria bone tissues. Further, they are induced by the TGF-β family. To analyze the expression of these matrices in pulp cells induced by TGF-β3, we performed immunostaining with the anti-OCN or anti-COL I antibody, and also examined the results with RT-PCR assays. Tooth germs were cultured under a serum-free condition. We observed immuno-positive reactions for OCN (Fig. 4A,b,c) and COL I (Fig. 4B,b,c), which accumulated around beads soaked with 10 ng/µL and 100 ng/µL of TGF-β3 in a dose-dependent manner, which was in contrast to the PBS-soaked beads (Fig. 4A,a,B,a). The intensity of the immunoreactions for OCN and COL I induced by the TGF-β3 beads was determined as described in Materials and Methods. As seen with the morphometry analysis method, OCN immunoreactivity and COL I immunoreactivities were increased by TGF-β3 in a dose-dependent manner (Fig. 4C). To confirm the expression of OCN and type I collagen, we performed real time RT-PCR assays with specific primer sets using mRNA isolated from tooth germ organ cultures. Exogenously administrated TGF-β3 significantly elevated the mRNA expression of both OCN and type I collagen, which was dependent on the concentration of the added TGF-β3 (Fig. 4D). These results suggest that TGF-β3 is a factor in the upregulation of the expression of both mRNA and proteins of OCN, as well as those of COL I (Table 1). As a control, a concomitant PCR assay for mouse β-actin was performed for each RT reaction.

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Figure 4. TGF-β3 induced the expression of OCN and COL I. We observed immunopositive reaction for OCN (Ab,Ac) and COL I (Bb,Bc), which accumulated around beads soaked with 10 ng/mL and 100 ng/mL TGF-β3 in a dose-dependent manner, in contrast to the PBS-soaked beads (Aa,Ba). Intensity of immunopositive area showed the dose-dependent increases for OCN (131 ± 1.1 for control, 202 ± 25.1 for PBS control, 388 ± 59.4 for the 10 ng/µL bead and 1267 ± 126.9 for the 100 ng/µL TGF-β3 one, N = 5), and for COL I (606 ± 42.2 for control, 637 ± 58.9 for PBS control, 825 ± 35.7 for the 10 ng/µL TGF-β3 bead and 1718 ± 144.6 for the 100 ng/µL TGF-β3 one, N = 5), respectively (C). Exogenously administrated TGF-β3 significantly elevated the mRNA expression of OCN (0.29 ± 0.04 for intact control, 0.31 ± 0.03 for PBS control, 1.00 ± 0.03 for 10 ng/mL TGF-β3 and 1.48 ± 0.07 for 100 ng/mL TGF-β3, N = 5), and COL I (0.26 ± 0.03 for intact control, 0.29 ± 0.03 for PBS control, 0.50 ± 0.04 for 10 ng/mL TGF-β3 and 1.14 ± 0.15 for 100 ng/mL TGF-β3, N = 5), respectively. These expression levels were dependent on the concentration of TGF-β3 (D). * = P < 0.05 compared with other groups in both C and D. Bars, 25 µm.

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Table 1.  Increased level of OCN, COL 1 and DSP: Comparison of one group to others (in fold)
 TGF100/IntactTGF100/PBSTGF100/TGF10TGF10/IntactTGF10/PBS
  1.  We detected a 9.7- and 6.3-fold increase of OCN and 2.8- and 2.7-fold increase of COL I immunoreactivities in 100 ng/µL TGF-β3 bead compared with each of the intact control, as well as the PBS bead; and 3.3-fold increase of OCN and 2.1-fold increase of COL I immunoreactivities when compared 100 ng/µL TGF-β3 bead to 10 ng/µL TGF-β3 bead. There was 3.0- and 1.9-fold increase of OCN and 1.4- and 1.3-fold increase of COL I immunoreactivities in TGF-β3 10 ng/µL bead compared to each of the intact control, as well as the PBS bead.

  2.  We detected 5.1- and 5.0-fold increase of OCN, 4.4- and 3.9-fold increase of COL I, 2.8- and 2.4-fold increase of DSP mRNA in 100 ng/mL TGF-b3 compared with each of the intact control, as well as PBS control; and 1.5-fold increase of OCN, 2.3-fold increase of COL I and 1.4-fold increase of DSP mRNA compared TGF-β3 100 ng/mL culture to TGF-β3 10 ng/mL culture. There was 3.4- and 3.2-fold increase of OCN, 1.9- and 1.7-fold increase of COL I, 2.0- and 1.7-fold increase of DSP mRNA in TGF-b3 10 ng/mL culture compared to each of intact control, as well as PBS control culture. All comparison results are significant (P < 0.05).

Protein
OC9.76.33.33.01.9
COL I2.82.72.11.41.3
mRNA
OC5.15.01.53.43.2
COL I4.43.92.31.91.7
DSP2.82.41.42.01.7

TGF-β3 induced mineralized nodule formation in cultured dental mesenchymal cells

Transforming growth factor-β3 was exogenously added to the dental mesenchymal cell cultures at a concentration of 10 or 100 ng/mL, to analyze the mineralization potential in vitro (Fig. 5). Primary cultures of dental mesenchymal cells derived from E17 tooth germ samples were gathered after 7 days of culture to form mineralized nodules (Fig. 5A,a). Alizarin Red S positive staining was observed in those cultured with 10 ng/mL TGF-β3 (Fig. 5A,c) and 100 ng/mL TGF-β3 (Fig. 5A,d). Whereas little or no staining was seen without TGF-β3 (Fig. 5A,b). The OD value of Alizarin Red S in cultured pulpal cells was analyzed according to the method described in Materials and Methods (Fig. 5B). We detected a 2.3-fold increase (P < 0.05) of mineralization in cultured pulpal cells with 100 ng/mL of TGF-β3 as compared with the control, and a 1.58-fold increase (P < 0.05) when compared to those with 10 ng/mL of TGF-β3 added. In addition, there was a 1.46-fold increase (P < 0.05) in the cultures with 10 ng/mL of TGF-β3 added as compared with the control. These results suggest that TGF-β3 induces mineralized tissue formation in vitro.

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Figure 5. TGF-β3 induced mineralized nodule formation in dental mesenchymal cells in vitro. Primary cultures of dental mesenchymal cells were gathered after 7 days culture to form a mineralized nodule (Aa). Alizarin Red S positive staining was observed in those cultured with 10 ng/mL TGF-β3 (Ac) and 100 ng/mL TGF-β3 (Ad), whereas little or no staining was seen without TGF-β3 (Ab). The optical density value of Alizarin Red S showed the dose-dependent increases of mineralized nodule (0.193 ± 0.053 for intact control, 0.202 ± 0.048 for PBS control, 0.294 ± 0.024 for 10 ng/mL, 0.465 ± 0.107 for 100 ng/mL, N = 5) (B). * = P < 0.05 compared with control group. Bars, 20 µm.

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DSPP and DMP1 expression in dental mesenchymal cells induced by TGF-β3

To identify the odontogenic progenitor population among the dental mesenchymal cells cultured with TGF-β3, we performed real time RT-PCR assays with specific primer sets for odontoblastic markers. The expressions of ONC, OPN and OCN, in both dental mesenchymal cells and calvaria osteoblasts, were increased after treatment with TGF-β3 (Fig. 6A). Further, DSPP and DMP1, which are expressed specifically in odontoblasts, were increased by 1.8-fold (P < 0.05) and 1.6-fold (P < 0.05), respectively, in dental mesenchymal cells following the addition of TGF-β3, whereas there was no increase detected in calvaria osteoblasts.

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Figure 6. DSPP and DMP1 expression in dental mesenchymal cells induced by TGF-β3. Expression of ONC, OPN and OCN in both dental mesenchymal cells and calvaria osteoblasts were increased after treatment with TGF-β3. Further, DSPP and DMP1, which are expressed specifically in odontoblasts, were increased by 1.8-fold (P < 0.05) and 1.6-fold (P < 0.05), respectively, in dental mesenchymal cells following the addition of TGF-b3, whereas there was no increase detected in calvaria osteoblasts (A). In situ hybridization analysis showed that there was no DSP message expression around the PBS bead (Ba). Positive staining for DSP was detected around the TGF-β3 soaked bead (Bb). Supplementation of exogenous TGF-β3 showed the dose-dependent increases of DSP message expression (0.78 ± 0.09 for intact control, 0.89 ± 0.07 for PBS control, 1.52 ± 0.09 for 10 ng/mL TGF-β3, 2.16 ± 0.17 for 100 ng/mL TGF-β3, N= 5) (C). * = P < 0.05 compared with other groups. Bars, 10 µm.

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To analyze the inductive expression of DSP in vivo, we performed in situ hybridization with tooth germ organ cultures. When cultured in the presence of PBS-soaked beads, there was no DSPP mRNA expression in the pulp region (Fig. 6B,a), whereas DSPP positive cells were detected in the pulp surrounding the TGF-β3-soaked beads (Fig. 6B,b). Further, DSPP mRNA was found expressed in odontoblasts in the intact control group, however, not in the dental pulp, enamel and ameloblast samples (data not shown). RT-PCR analysis using mRNA from organ cultures showed that TGF-β3 significantly induced the expression of DSPP in a dose-dependent manner (Fig. 6C; Table 1). Thus, our results indicated that TGF-β3 is a factor involved in the upregulation of dentin specific gene expression in dental pulp cells in vitro and in vivo.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dental pulp cells consist mainly of fibroblasts as well as dentin pulp stem cells (Gronthos et al. 2000), both of which have been suggested to play roles in dental pulp tissue healing and repair. It is widely accepted that these cells can differentiate into odontoblast-like cells and form reparative dentin (Yamamura 1985); however, the factors that control the process of pulp tissue formation and repair are not clearly understood. During mouse tooth development, TGF-β1 is initially expressed in the oral epithelium on E13, while later its expression extends into the mesenchymal compartment and then becomes restricted to the ectomesenchyme layer in odontoblasts. This odontoblast-restricted expression of TGF-β1 then persists throughout life in mice (Vaahtokari et al. 1991).

Transforming growth factor-β receptors II and I are expressed in odontoblasts and pulp cells in human teeth (Sloan et al. 2001); however, the expression of many types of receptors for TGF-β in rat incisor pulp and bovine adult pulp tissues indicates diverse functions in tooth development and pulp repair (Toyono et al. 1997a; Toyono et al. 1997b). In fact, TGF-β has been implicated to function as an inducer of dentin matrix formation during dentinogenesis, while it has also been shown to influence the differentiation of pulp fibroblasts and formation of reparative dentin in vivo (Hu et al. 1998). Further, in vitro experiments have shown that TGF-β is able to stimulate cell proliferation and matrix secretion, such as COL I and osteonectin/SPARC (secreted protein, acidic and rich in cysteine) (Nakashima et al. 1994; Shirakawa et al. 1994; Shiba et al. 1998).

On the other hand, teeth in mice that overexpress TGF-β1 show a significant reduction in tooth mineralization along with defective dentin formation, as dentin extracellular matrix components such as type I and III collagen, are increased and deposited abnormally in dental pulp, similar to the hereditary human tooth disorders such as dentinogenesis imperfecta (Thyagarajan et al. 2001). In these mice, DSPP, a candidate gene implicated in dentinogenesis imperfecta II, is significantly downregulated; thus, in vivo evidence suggesting that TGF-β1 mediates the expression of DSPP is crucial to understand the process of dentin formation. Similar results have been reported from in vitro experiments (He et al. 2004). TGF-β1-activated Smad signaling led to a Smad3-mediated downregulation of DSPP in an odontoblast cell line. However, the effect of TGF-β3 on pulp cells is not well understood, it has lower levels of expression as compared with TGF-β1 (Cassidy et al. 1997).

The present study is the first to show that TGF-β3 induces ectopic mineralization in dental pulp through upregulation of the expression of OCN and COL I. Further, the expression of DSPP in tooth germ cultures and primary cultured pulp cells was also increased in the presence of TGF-β3. The COL I expression induced by TGF-β3 is similar to the effect of TGF-β1 with pulp cells, as previously reported (Nakashima et al. 1994; Shirakawa et al. 1994; Shiba et al. 1998), whereas the expression of DSPP induced by TGF-β3 is quite different from that induced by TGF-β1, which may be a specific effect of TGF-β3 during odontoblast-like cell differentiation.

The TGF-β family is competent for the early stages of chondroblastic and osteoblastic differentiation; however, it inhibits late-stage osteoblast differentiation (Roelen & Dijke 2003). Others have reported that TGF-β3 is able to promote chondrogenic differentiation in cultures of human mesenchymal stem cells isolated from bone marrow (Mackay et al. 1998), or that it completely inhibited the process of redifferentiation in cultures of mouse embryonic stem (ES) cells (Hegert et al. 2002). The present findings, together with previous data, suggest that TGF-β3 induces mineralizing protein, COL I and OCN, which provides an organic framework for deposition of inorganic components such as calcium. A possible mechanism is that TGF-β3 stimulates the surrounding pulpal mesenchymal cells to secrete TGF-β3 in an autocrine manner, while processes within the cell itself are responsible for differentiation and extracellular matrix production.

Dentin matrix protein and DSP show characteristic temporal and spatial expression patterns within odontogenic tissue during the process of dentin mineralization (Hao et al. 2004). It has also been reported that the DSPP-null mice develop tooth defects similar to human dentinogenesis imperfecta III, with enlarged pulp chambers, an increased width of the predentin zone, hypomineralization and pulp exposure (Sreenath et al. 2003). DSP is a specific biochemical marker of functional odontoblasts because it is synthesized and secreted by differentiated odontoblasts (Ritchie et al. 1997; Narayanan et al. 2001). However, a recent study indicated that DSP is more widely distributed, such as in the alveolar bone, cementum and periodontal ligament (Baba et al. 2004). OCN plays a role in early stages of the process of dentin matrix mineralization, whereas DSP is involved during the more advanced steps (Bleicher et al. 1999). The present in situ hybridization results showed that DSP mRNA was expressed in some pulp cells around the implanted beads soaked with TGF-β3, which suggests that TGF-β3 stimulates the differentiation of pulp cells with DSP expression and increases the biosynthetic activity to induce mineralized tissue. Interestingly, the major part of the dental pulp tissue around TGF-β3-soaked beads did not express DSP, indicating that the tissues induced by TGF-β3 in the present study may have been in the early stages of mineralization. Further investigation should provide a better understanding to completely define the character of mineralized tissue induced by TGF-β3.

The expression of the osteoblastic markers, osteonectin, osteopontin and OCN were increased following TGF-β3 treatments in both dental pulp cells and osteoblasts in the present study, indicating that TGF-β3 regulates osteoblast phenotypes as a promoter. DSPP and DMP1 are expressed in both osteoblasts and dental pulp cells; however, the expression levels in osteoblasts are lower than in dental pulp cells. Although it has been reported that TGF-β1 downregulated both DMP1 and DSPP genes in a mouse odontoblast cell line (Unterbrink et al. 2002), the expression of DSPP and DMP1 were dramatically increased after treatment with TGF-β3 in dental papilla cells, but not in osteoblasts, indicating that TGF-β3 is important for odontoblast differentiation. In the present in vivo experiments, ectopic administration of TGF-β3 induced bone like tissues without a dentinal tube-like structure around the implanted beads. These results indicate that TGF-β3 may induce odontoblast-like cells in dental pulp, though other factors may be also involved in this step.

Stem cells in dental pulp may be mobilized to differentiate into odontoblasts by morphogens released from the surrounding dentin matrix (Tziafas et al. 2000). Further, dental pulp tissue has the potential to regenerate dentin in response to noxious stimuli, such as caries, and progenitor/stem cells are responsible for this action. Thus, stem cell therapy shows considerable promise for dentin regeneration therapy (Iohara et al. 2004). Implantation of a bead with biologically active molecules may be a suitable model for study of the molecular events involved with tooth development.

In summary, we showed for the first time the role of TGF-β3 in inducing ectopic mineralization in fetal mouse pulpal tissues. Possible mechanisms of this TGF-β3 function include modulating the matrix composition, composed of COL I, OCN and DSPP, and promoting the differentiation of pulp mesenchymal cells to induce ectopic mineralization.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Yukiko Abe, Nagasaki University, for her technical support. The present study was supported in part by Grants-in-Aid to S. SUITA (16390504), K. Nonaka (15390638) and M. Ohishi (14370677), from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. This manuscript is based on a thesis submitted by M. Huojia to the Graduate School of Dental Science, Kyushu University, in partial fulfillment of the requirements for a PhD degree.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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