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J Dent Res 86(5):469-474, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Hard Tissue Formation in Subcutaneously Transplanted Rat Dental Pulp

A. Hosoya1,*, H. Nakamura1, T. Ninomiya2, K. Hoshi3, K. Yoshiba4, N. Yoshiba4, M. Takahashi5, T. Okabe6, N. Sahara2, H. Yamada7, E. Kasahara7, and H. Ozawa2

1 Department of Oral Histology, Matsumoto Dental University, 1780 Gobara Hirooka, Shiojiri, Nagano 399-0781, Japan;
2 Institute for Dental Science, Matsumoto Dental University, Nagano, Japan;
3 Department of Menicon Cartilage and Bone Regeneration, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;
4 Division of Cariology, Operative Dentistry and Endodontics, Department of Oral Health Science, Course for Oral Life Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;
5 Division of Cardiovascular Science, Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Nagano, Japan;
6 Department of Orthopedic Surgery, Shinshu University Graduate School of Medicine, Nagano, Japan; and
7 Department of Endodontics and Operative Dentistry, Matsumoto Dental University, Nagano, Japan

* corresponding author, hosoya{at}po.mdu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
While dental pulp appears to be able to form mineralized matrices that do not always resemble dentin, the precise characteristics of the hard tissue and the mechanism of its induction remain unknown. Therefore, we evaluated hard tissue induced by transplantation of pulp into subcutaneous tissue. Seven days after transplantation, initial hard tissue was formed at the inner periphery of the pulp. After 14 days, this hard tissue expanded inwardly. Mineralized matrix was immunopositive for osteocalcin, osteopontin, and bone sialoprotein, but negative for dentin sialoprotein. Transplantation of GFP-labeled pulp into wild-type rats showed these formative cells to have been derived from the transplant. TEM observation revealed apatite crystals within necrotic cells and matrix vesicles at the initial stage of calcification. These results indicate that pulp cells possess the ability to form a bone- or cementum-like matrix. Calcification of the matrix may occur in necrotic cells and matrix vesicles, followed by collagenous calcification.

KEY WORDS: dental pulp calcification • transplantation • non-collagenous proteins • rat incisor • immunohistochemistry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dental pulp is involved in reparative dentin and dentin bridge formation in response to a variety of external stimuli. Several morphological studies have suggested that dental pulp is capable of hard tissue formation. Calcification of pulp was observed after trauma (Robertson et al., 1997), tooth replantation (Ohshima et al., 2001), tooth transplantation into subcutaneous tissue (Hosoya et al., 2003), and pulpal exposure of rat teeth (Tsuji et al., 1987). However, these reports evaluated pulp calcification in the presence of dentin matrix. Dentin contains several growth factors, such as bone morphogenetic proteins and transforming growth factors (Butler et al., 1977; Bessho et al., 1991). These growth factors promote odontoblastic differentiation and calcified matrix formation in vivo (Hu et al., 1998; Smith and Lesot, 2001) and in vitro (Sloan et al., 2000). Thus, it is unclear whether pulp calcification is caused by the pulp cells themselves or induced by growth factors in dentin.

In contrast, several reports demonstrated that isolated rat incisor pulp could induce calcified tissue, which did not resemble dentin, via transplantation into subcutaneous tissue, kidney capsule, or the anterior chamber of the eye (Yamamura, 1985; Inoue and Shimono, 1992; Yamazoe et al., 2002). However, the origin of these hard-tissue-forming cells and the precise characteristics of these tissues remain unknown. In the present study, we examined the process of induction of mineralized tissue by subcutaneously transplanted rat incisor pulp in the absence of dentin. This model enabled us to investigate the mechanism of pulp calcification without the effects of growth factors in dentin. The matrix composition was examined immunohistochemically by the use of osteocalcin (OCN)-, osteopontin (OPN)-, bone sialoprotein (BSP)-, and dentin sialoprotein (DSP)-specific antibodies. The calcified matrix was also analyzed histologically by transmission electron microscopy (TEM).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Preparation
All experiments were performed according to strict guidelines set forth by the Matsumoto Dental University Committee on Intramural Animal Use. Seventy male wild-type or green fluorescent protein (GFP)-transgenic rats, 6 wks of age, were used in this experiment. After the animals were anesthetized, both mandibles were removed, and alveolar bone was then dissected. The pulp tissue was then isolated from the incisors and immersed in sterilized physiological saline. The middle portion of the pulp core, an approximately 2.5- to 3-mm3 mass, was transplanted into a pre-formed subcutaneous pouch in the head of each host rat. At 3, 7, and 14 days after transplantation, the transplanted pulps with surrounding tissue were removed and fixed. For back-scattered electron (BSE) analysis and ultrastructural observations, some specimens were fixed with 1% acrolein, 2% paraformaldehyde, and 3% glutaraldehyde in 0.04 M cacodylate buffer (pH 7.4) at 4°C for 20 hrs. Other samples were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) for light and immunoelectron microscopy. Specimens were obtained from 10 rats at each time period. As a control, 4 freshly isolated pulp samples were fixed so that the state prior to transplantation could be observed.

Lewis transgenic rats, which ubiquitously express enhanced GFP (Hakamata et al., 2001; Inoue et al., 2005), were a generous gift from YS New Technology Institute (Tochigi, Japan). To clarify the origin of hard-tissue-forming cells, we transplanted some pulps of GFP-transgenic rats into the subcutaneous tissue of wild-type rats, and processed them in the same manner as described above.

Back-scattered Electron (BSE) Analysis
To investigate the degree of calcification, we obtained BSE images by using an energy-dispersive x-ray spectrometer (JSM-6360LA; JEOL Ltd., Tokyo, Japan). After fixation with 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) for 1 hr at 4°C, undecalcified specimens were embedded in Epon 812 (TAAB, Berkshire, England). The samples were cut approximately in half, and carbon-coated (JEE-420; JEOL Ltd., Tokyo, Japan) to prevent charging. We measured areas of calcified pulp by using a beam current of 60 nA and an accelerating voltage of 16 kV.

Light Microscopy
The specimens were demineralized with 5% EDTA (pH 7.4) for 2 wks at 4°C, and then embedded in paraffin. Thin sections (4-µm thickness) were stained with hematoxylin and eosin (H-E). Serial sections were also processed for immunohistochemistry with polyclonal goat antibodies against rat OCN (Biomedical Technologies, Stoughton, CA, USA) and polyclonal rabbit antibodies against mouse OPN (provided by Dr. M. Fukae), human BSP (LF-120, provided by Dr. L.W. Fisher), rat DSP (provided by Dr. W.T. Butler), and GFP (Molecular Probes Inc., Eugene, OR, USA). Immunostaining was performed by the avidin-biotin-complex method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA). In these procedures, the antibodies against OCN, OPN, BSP, and GFP were diluted to 1:500, and DSP antibodies to 1:1000. In addition, DSP-stained sections were subjected to microwave energy while immersed in 10 mM citric acid buffer (pH 6.0) at 70°C for 10 min before these procedures for antigen retrieval. The immune complexes were visualized by the use of diaminobenzidine (Envision kit; DAKO, Carpinteria, CA, USA), and the sections were counterstained with hematoxylin. As positive controls for these antibodies, sections of dentin in a normal molar tooth were immunostained. In addition, non-immune goat or rabbit sera were used in place of the primary antibodies, as negative controls.

For detection of cell proliferation, several animals were intraperitoneally injected with bromodeoxyuridine (BrdU; Sigma, St. Louis, MO, USA) at a dosage of 2.5 mg/100 g body weight 1 hr before fixation. After pre-treatment with 2 N HCl for 30 min at 37°C, followed by 0.1% trypsin in phosphate-buffered saline (PBS, pH 7.4) for 15 min at room temperature, sections were incubated with 1:1000 diluted monoclonal antibody against BrdU (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) for detection of the BrdU. Immunoreactivity was visualized with Vectastain Elite ABC and Envision kits.

Transmission Electron Microscopy
Undecalcified and decalcified samples were post-fixed with 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) for 1 hr at 4°C, and embedded in Epon 812. Ultrathin sections were cut by means of an ultramicrotome (Ultracut UCT; Leica Instruments, Wetzlar, Germany), and the decalcified sections were stained with uranyl acetate and lead citrate. The undecalcified sections were collected on ethylene glycol and left unstained. These sections were observed by TEM (H-7600; Hitachi Co., Tokyo, Japan) at an accelerating voltage of 80 kV.

Immunoelectron Microscopy
The decalcified specimens were embedded in LR White Resin (London Resin Company Ltd., London, England). After pre-treatment with 10% bovine serum albumin (Seikagaku, Tokyo, Japan) in PBS, ultrathin sections were incubated with antibodies against OPN or BSP (1:200) for 12 hrs at 4°C. The sections were then rinsed in PBS and reacted with 10 nm gold-conjugated goat antibodies against rabbit IgG (1:50; Amersham Biosciences, Buckinghamshire, UK) for 1 hr at room temperature. They were stained with 1% tannic acid and uranyl acetate. After having been supported with formvar film, the sections were observed by TEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BSE Analysis
Three days following transplantation, no calcified area was detected by BSE analysis (not shown). However, at 7 days, areas of calcification were observed on the inner boundary of the pulp. These areas showed a globular appearance. Some globules had coalesced to form large areas. By 14 days, widespread calcification was seen throughout the transplanted pulp, although a few uncalcified areas remained inside the pulp (Figs. 1a–1cGo).


Figure 1
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  		Figure 1. BSE analysis (a–c) and light micrographs of sections stained by H-E (d–f) or immunostained for GFP (g,h) or BrdU (i–l). Transplanted pulp after 3 (d,i), 7 (a,e,j), and 14 (b,f,g,h,k) days. (a) Calcified hard tissue of the inner boundary of the pulp is evident. (b) Most of the pulp tissue is calcified. (c) The area of mineralized tissue was measured from images obtained by BSE analysis. (d) Typical odontoblasts have disappeared (asterisks) in the periphery of the pulp. (e) Extracellular matrix (stars in e–g,j,k) has formed within the peripheral pulp. (f) Matrix formation extends into the pulp. (g,h) GFP-positive cells are detected within the matrix and on its surface. "h" is an enlargement of the area denoted by the rectangle in "g", with the arrowheads pointing to GFP-positive cells on the surface of the matrix. (i) BrdU-positive cells are not seen in the pulp. (j) Numerous positive cells are apparent in the center of the pulp. (k) Positive cells have decreased in number. (l) Number of BrdU-positive cells counted in the immunostained sections. (c,l) Data are expressed as the means ± SE from 5 specimens per group. ANOVA and Student’s t test show a significant difference between the 2 groups. p values < 0.05 are considered statistically significant. The boundary between the transplant and the surrounding connective tissue (CT) is indicated by the dotted line (d–g, i–k). BV, blood vessel. Scale bar: 300 µm (a,b), 120 µm (g, i–k), 100 µm (d–f), 40 µm (h).

 
Histological Observations
The removed pulp did not contain dentin matrix, although the odontoblast layer remained (not shown). Three days after transplantation, the transplanted pulp was surrounded by granulation tissue. Most of the odontoblasts had degenerated, as evidenced by the disappearance of cuboid cells. Numerous fibroblast-like cells were seen in the pulp (Fig. 1dGo). At 7 days, eosin-stained extracellular matrix containing numerous cells was observed in the transplant, into which blood vessels had invaded (Fig. 1eGo). By 14 days, matrix formation had expanded inwardly, and large round cells had aligned on the matrix. However, the matrix did not contain any tubular structures (Fig. 1fGo; Appendix Fig., a). All 6 samples at each time period showed a similar appearance. Inflammatory cells, such as neutrophils and lymphocytes, were not seen at any stage after transplantation.

Immunohistochemical Observations
At 14 days, GFP-positive cells were detected within the newly formed matrix. The lining cells on the matrix and the pulp cells were also immunopositive for GFP (Figs. 1g, 1hGo).

BrdU-positive cells, indicating DNA synthesis, were not observed in the transplanted pulp at 3 days. At 7 days, numerous BrdU-positive cells were seen in the center of the transplanted pulp. The number of BrdU-positive cells declined at 14 days (Figs. 1i–1lGo).

Localization of OCN, OPN, and BSP was detected in the newly formed matrix. Immunoreactivity was seen in globular aggregates of the matrix (Figs. 2a–2cGo), corresponding to the mineralized area (Appendix Fig., b). However, DSP was not detected there (Fig. 2dGo). In contrast, the dentin matrix was immunopositive for OCN, OPN, BSP, and DSP (Figs. 2e–2hGo). The staining controls did not show any specific immunoreactivity (Figs. 2i, 2jGo).


Figure 2
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  		Figure 2. Immunohistochemical staining for OCN (a), OPN (b), BSP (c), and DSP (d) in the pulp 14 days after transplantation, positive controls (eh) for immunohistochemical staining for OCN (e), OPN (f), BSP (g), and DSP (h), and negative controls (i,j). (a–c) OCN, OPN, and BSP are localized within newly formed tissue, where the immunoreactivity is detected in globular aggregates. (d) DSP immunoreactivity is undetectable within the matrix. (e–h) Sections of a six-week-old normal molar tooth. OCN, OPN, and BSP immunoreactivities are detected in dentin matrix (D). Additionally, DSP is localized heavily in the dentin. (i,j) Control sections of pulp 14 days after transplantation. No immunoreaction is seen with non-immune goat or rabbit serum. Scale bar: 30 µm (a–j).

 
Transmission Electron Microscopy
At 14 days, cells in 2 different states were seen in the newly formed matrix. One was characterized by having a well-developed Golgi apparatus and rough-surfaced endoplasmic reticulum (rER). The other was necrotic and contained numerous vacuoles. Several vesicular structures were also observed in the extracellular matrix (Fig. 3aGo). Meanwhile, in the central region of the pulp, cells lying in the newly formed matrix contained a highly developed Golgi apparatus and rER. Matrix vesicles were observed in the matrix under these cells (Fig. 3bGo, arrows in the inset).


Figure 3
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  		Figure 3. Electron micrographs of internal (a) and superficial (b) hard tissue in the 14-day transplant. (a) Numerous vesicle-like structures (arrows in the insert) are present in the extracellular matrix. An intact cell (asterisk) containing well-developed cellular organelles is also seen. (b) Matrix vesicles (arrows in the insert) are observed in the extracellular matrix. Go, Golgi apparatus; N, nucleus; rER, rough-surfaced endoplasmic reticulum. Scale bar: 2 µm (a,b), 0.2 µm (inserts of a,b).

 
Undecalcified ultrathin sections revealed the calcification process in the subcutaneously transplanted pulp. At initial calcification sites, vesicle-like structures containing apatite crystals were observed in the necrotic cells (Fig. 4aGo). Similar apatite crystals were also observed in the extracellular matrix surrounding degenerated cells (Fig. 4bGo). In regions of advanced calcification, calcified nodules came into contact with collagen fibrils (Fig. 4cGo), and large areas of calcified hard tissue were formed during the progress of collagenous calcification (Fig. 4dGo).


Figure 4
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  		Figure 4. Electron micrographs demonstrating various stages of calcification (a–d) and OPN localization (e,f) after 7 days. (a,b) At initial calcification sites, apatite crystals are observed within and outside the cells. (c,d) Calcification progresses to collagen fibrils (arrows in "c") from calcified nodules (CN). (e) Immunoreactivity for OPN is seen in the peripheral region of a calcified nodule. (f) The expanded collagen fibrils (asterisks) are immunopositive for OPN. Scale bar: 0.5 µm (d,e,f), 0.2 µm (b,c), 0.1 µm (a).

 
Immunoelectron Microscopy
Gold particles representing OPN localization were detected in the periphery of calcified nodules (Fig. 4eGo). As calcification advanced, OPN immunoreactivity was observed within collagen fibrils (Fig. 4fGo). Immunolocalization of BSP showed a pattern similar to that for OPN (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, subcutaneous transplantation of pulp induced pulp calcification without inflammation and rejection. All transplanted pulp specimens demonstrated similar progress of calcification. Since the transplanted pulp had been separated from the surrounding dentin, we were able to observe the process of hard tissue formation by pulp tissue without the influence of dentin. Therefore, this model is a useful and reliable method for assessing the mechanism of pulp calcification.

OCN, OPN, and BSP are present in various hard tissues, such as bone, dentin, and cementum (Chen et al., 1993; Bronckers et al., 1994; MacNeil et al., 1995). In contrast, DSP has been reported to be a highly specific matrix protein for dentin (D’Souza et al., 1992). In the transplanted pulp, the newly formed calcified tissue was immunopositive for OCN, OPN, and BSP, but not for DSP. This pattern was similar to that seen in bone and cementum. In addition, the calcified matrix contained numerous cells. No dentinal tubular structures, characteristic of dentin, were seen in the matrix. Furthermore, the hard-tissue-forming cells in the matrix resembled osteoblasts and cementoblasts, rather than odontoblasts. Osteodentin formed after cavity preparation and dental pulp-capping has been reported to be immunopositive for DSP (D’Souza et al., 1995; Andelin et al., 2003). Thus, the hard tissue formed in subcutaneously transplanted pulp is different from dentin or osteodentin. Tooth development is initiated by reciprocal interactions between epithelial and mesenchymal cells. Mesenchymal cells differentiate into odontoblasts, which secrete dentin matrix (Nanci, 2003). In the present model, the formation of bone- or cementum-like hard tissue by pulp cells may have resulted from the absence of epithelium and/or dentin matrix. Epithelial cell signals and/or dentin-derived growth factors might be required for the formation of dentin matrix, and pulp cells do not appear to be able to substitute for this inductive influence.

At 7 and 14 days after transplantation, blood vessels were seen adjacent to the newly formed hard tissue. The blood vessels could form the environment for mineralization events by secreting active molecules. In contrast, it has been described earlier that subcutaneously transplanted rabbit muscle formed cartilage (Bridges and Pritchard, 1958). Additionally, perichondrium and periosteum transplants appeared to induce cartilage and bone, respectively (Yamamura, 1985). Therefore, the origin of the transplanted tissue might determine the type of hard tissue in contact with the surrounding environment produced by blood vessels.

In the process of hard tissue formation, initial apatite crystals were observed within necrotic cells. It has been suggested that cell debris might provide a nucleus for calcification following the subcutaneous injection of calcium hydroxide (Yoshiba, 1988). A similar mechanism might occur in dentin bridge formation, following direct pulp-capping with calcium hydroxide, since a necrotic layer is always in contact with the dentin bridge. Moreover, the degenerated pulp has a high incidence of pulp stone formation. These findings suggest that necrotic cells are involved in the initiation of calcification. Although pulps did not calcify while they were in the tooth under physiological conditions, they did become calcified immediately after having been transplanted subcutaneously. Furthermore, in the present study, necrotic pulp cells became calcified and then surrounded by newly formed hard tissue. These results suggest that degenerated odontoblasts and necrotic pulp cells might provide nucleation sites for apatite crystal formation.

In this study, we found that the matrix formed in the transplanted pulp contained OPN and BSP. OPN and BSP are known to exist in calcified nodules in osteoid (McKee et al., 1993, Hoshi et al., 2001). Although BSP possesses nucleation activity toward hydroxyapatite in vitro, OPN inhibits crystal growth (Hunter et al., 1996). Our electron microscopic immunocytochemical results, showing that OPN and BSP were localized in calcified nodules and collagen fibrils in contact with the nodules, suggest that OPN and BSP might play a role in regulating crystal growth and formation.

The experiment with GFP-labeled rats revealed that the hard-tissue-forming cells indeed originated from the pulp cell population. Cells in the odontoblast layer were necrotic and formed initial apatite crystals at 7 days after transplantation. We also observed that BrdU-positive cells were seen in the central region of the pulp, suggesting that undifferentiated cells in the pulp might have proliferated and differentiated into calcified matrix-forming cells. Hence, the initial apatite crystals within necrotic cells might have acted as a trigger for the differentiation of these hard-tissue-forming cells. The existence of stem cells within dental pulp has been reported in human permanent (Gronthos et al., 2000) and deciduous (Miura et al., 2003) teeth. Pulp tissue contains mesenchymal cells that can differentiate into hard-tissue-forming cells.

In conclusion, subcutaneously transplanted rat dental pulp formed mineralized hard tissue possessing bone- or cementum-like characteristics. Our findings thus indicate that pulp cells are able to form mineralized hard tissue in the absence of dentinal growth factors, but the morphology of the tissue formed does not resemble that of dentin.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Fukae (Tsurumi University School of Dental Medicine), Dr. L.W. Fisher (National Institute of Dental and Craniofacial Research, US National Institutes of Health), and Dr. W.T. Butler (University of Texas-Houston Health Science Center) for kindly providing the antibodies against OPN, BSP, and DSP, respectively. We are also grateful to YS New Technology Institute for providing GFP-transgenic rats, and to S. Akahane, H. Komatsu, and H. Koike (Matsumoto Dental University) for their technical support. This work was supported by grants-in-aid for scientific research (Nos. 16659506 and 17791372) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received April 21, 2005; Last revision November 15, 2006; Accepted December 23, 2006


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 RESULTS
 DISCUSSION
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