| Sign In to gain access to subscriptions and/or personal tools. |
Dentin Regeneration by Dental Pulp Stem Cell Therapy with Recombinant Human Bone Morphogenetic Protein 2
1 Department of Clinical Oral Molecular Biology, Division of Oral Rehabilitation, Correspondence: * corresponding author, misako{at}dent.kyushu-u.ac.jp
Regenerative medicine is based on stem cells, signals, and scaffolds. Dental pulp tissue has the potential to regenerate dentin in response to noxious stimuli, such as caries. The progenitor/stem cells are responsible for this regeneration. Thus, stem cell therapy has considerable promise in dentin regeneration. Culture of porcine pulp cells, as a three-dimensional pellet, promoted odontoblast differentiation compared with monolayers. The expression of dentin sialophosphoprotein (Dspp) and enamelysin/matrix metalloproteinase 20 (MMP20) mRNA confirmed the differentiation of pulp cells into odontoblasts and was stimulated by the morphogenetic signal, bone morphogenetic protein 2 (BMP2). Based on the in vitro experiments, an in vivo evaluation of pulp progenitor/stem cells in the dog was performed. The autogenous transplantation of the BMP2-treated pellet culture onto the amputated pulp stimulated reparative dentin formation. In conclusion, BMP2 can direct pulp progenitor/stem cell differentiation into odontoblasts and result in dentin formation.
Key Words: dentin regeneration • stem cell therapy • BMP2 • dental pulp-capping • pellet culture Abbreviations: BMP2, bone morphogenetic protein 2 • Dspp, dentin sialophosphoprotein • Dmp1, dentin matrix protein 1 • ALPase, alkaline phosphatase • MMP20, matrix metalloproteinase 20 • Phex, phosphate-regulating gene with homologies to endopeptidases on X-chromosome
The dental pulp contains progenitor/stem cells, which can proliferate and differentiate into dentin-forming odontoblasts (Nakashima et al., 1994; Gronthos et al., 2000, 2002). Damaged odontoblasts can be replaced by newly generated populations of odontoblasts derived from stem cells from pulp (Tziafas et al., 2000). Following physiological stimulation or injury, such as caries and operative procedures, stem cells in pulp may be mobilized to proliferate and differentiate into odontoblasts by morphogens released from the surrounding dentin matrix (Tziafas et al., 2000). Tissue engineering with the triad of dental pulp progenitor/stem cells, morphogens, and scaffolds may provide a useful alternative method for pulp-capping and root canal treatment (Nakashima and Reddi, 2003). In pulp cell therapy, the technique for manipulation of the growth of the isolated pulp progenitor/stem cells and induction of three-dimensional tissue formation in vitro needs to be developed. Naturally derived collagen or synthetic materials such as polyglycolic acid (PGA) are used as a scaffold for attachment and guidance of cells (Putnam and Mooney, 1996). The pulp-derived fibroblasts adhering to the PGA fibers can proliferate and form a new tissue similar to that of native pulp (Mooney et al., 1996). The synthetic matrices, however, must undergo degradation simultaneously with the new tissue formation by the cultured cells. An alternative to the use of synthetic matrix with variable degradation rate is the use of three-dimensional cultures with an assembly of endogenous extracellular matrix or scaffold. A three-dimensional in vitro culture system has been developed for chondrocytes (Kato et al., 1988; Ballock and Reddi, 1994), bone marrow stromal cells (Yoo et al., 1998), and intervertebral disc cells (Lee et al., 2001). This system involves formation of cell pellets or aggregates by a one-step centrifugation method, allowing for three-dimensional interaction between the neighboring cells, and is followed by synthesis of extracellular matrix in the pellet. Cell-cell interactions and environmental cues are important in modulation of the phenotype of cells grown in vitro. Bone morphogenetic proteins (BMPs) have been implicated in tooth development, and the expression of BMP2 is increased during the terminal differentiation of odontoblasts (Nakashima et al., 1994; Nakashima and Reddi, 2003). Beads soaked in human recombinant BMP2 induce the mRNA expression of Dspp, the differentiation marker of odontoblasts after implantation onto dental papilla in organ culture. BMP2 also induces a large amount of reparative dentin on the amputated pulp in vivo (Nakashima, 1994a). It has been suggested that BMP2 may regulate the differentiation of pulp cells into odontoblastic lineage and stimulate reparative dentin formation (Nakashima and Reddi, 2003). We have compared three-dimensional pellet culture system with monolayer cultures. The efficacy of BMP2 on the differentiation of pulp cells into odontoblasts was also examined with the use of this pellet culture system. In addition, we investigated cell therapy in vivo for dentin regeneration.
Three-dimensional Pellet Culture of the Pulp Cells The porcine premolar pulp was isolated, and the pulp cells were separated enzymatically as previously described (Nakashima, 1991). The pellet culture of the porcine pulp cells was then performed (Kato et al., 1988; Ballock and Reddi, 1994). Briefly, 1-mL aliquots containing 2 x 105 cells were centrifuged in a 15-mL conical polypropylene tube (Asahi techno glass corp., Tokyo, Japan) at 1000 rpm for 5 min. Pellets were maintained in Dulbecco’s modified essential medium (Life Technologies, Rockville, MD, USA) supplemented with 10% heat-inactivated bovine calf serum (JRH Biosciences, Lenexa, KS, USA) and 50 µg/mL L-ascorbic acid phosphate (Wako pure chemical industries, LTD, Osaka, Japan), and penicillin-streptomycin. The medium was changed two times per wk. From day 28 on, the medium was supplemented with Pi to a 1-mM final concentration. For comparison, a traditional monolayer culture was performed at a density of 1 x 105 cells/mL in a 35-mm dish. In some experiments, recombinant human BMP2 (10, 25, 50, 100, and 200 ng/mL) (kindly provided by Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan) was added to the medium 1 hr before centrifugation after enzymatic isolation.
Total Cell Number and Alkaline Phosphatase Activity
Tissue Morphology
Quantification of Collagen Type I and Type III Syntheses
Real-time RT-PCR
Transplantation of the Pellet on the Canine Amputated Pulp Twenty-four teeth from 6 young adult dogs were used. Surgical anesthesia was obtained in the dogs by intraveneous administration of pentobarbital sodium. The upper incisor pulp was extracted and autogenous pellets were prepared as described earlier for porcine pulp. A surgical exposure was made in the canine, and the amputation was carried out. Pellet cultures were applied to the amputated pulp on day 14, and the cavity was filled with glass-ionomer cement and composite resin. Four wks after transplantation, dentin formation was examined in the serial paraffin sections. Our animal use protocols (porcine and canine) were reviewed and approved by the Kyushu University Institutional Review Board.
Comparison of the Pellet Culture with the Normal Monolayer Culture First, the sequential morphological changes in the pellet were examined. The pulp cells were oval or polygonal, and the nuclei contained a few round or ovoid nucleoli. The pellet progressively became spherical on days 14 and 21. On day 14, the cell density decreased, the cells became basophilic, and the extracellular matrix accumulated. On day 21, pellet cultures contained cells surrounded by newly formed matrix, osteodentin. The cells were faintly basophilic, and their nuclei were round or oval and darkly staining (Fig. 1A  ).
In general, there was minimal cell proliferation in pellet cultures compared with monolayers as enumerated by cell numbers (Fig. 1B  ). In contrast, the cells in the monolayer culture proliferated. The alkaline phosphatase activity gradually increased and was significantly higher on day 10 and day 14 in the pellet compared with the monolayer culture (Fig. 1C). The mRNA expression of  1(I)collagen was much higher on days 10 and 14 in pellet cultures than that in monolayer cultures, indicating extracellular matrix accumulation. The expression of Dmp1, Dspp, enamelysin, and Phex, differentiation markers, was much increased on day 21, compared with expression in the monolayer culture (Fig. 1D).
Effect of BMP2 on Differentiation and Mineralization in the Pellet Culture
Effect of BMP2 on Reparative Dentin Formation in the Pulp Cells Autogenous transplantation of the pellet was performed on the canine amputated pulp. Pulp cells labeled by transfection with adenovirus delivering CMV-lacZ cDNA before the pellet was made showed transgene lacZ expression in the pulp cells 14 days after transplantation (Figs. 3A , 3B), indicating successful transduction. We examined the feasibility of therapeutic reparative dentinogenesis using the pellet on the amputated pulp. In the control group, fibrodentin formation was not observed (Fig. 3C). Fibrous osteodentin matrix was observed in the cavity on the amputated pulp 4 wks after transplantation of the non-treated pellet (Fig. 3D). Tissue necrosis and inflammation were not observed. This suggested that the transplanted pellet was involved in reparative dentin formation in vivo. The osteodentinoblasts produced extracellular matrix around them (Fig. 3F). Large amounts of osteodentin were seen in the BMP2-treated pellet (Fig. 3E), compared with the non-treated pellet. A few osteodentinocytes were observed in the osteodentin (Fig. 3G). Odontoblast-like cells with long processes attached to the osteodentin to form tubular dentin (Fig. 3H).
Cell therapy utilizing pulp progenitor/stem cells has the potential to improve on conventional pulp-capping with calcium hydroxide or other artificial materials that can induce only a small amount of reparative dentin beneath the exposed or amputated site of the pulp. As a first step toward the goal of successful development of cell therapy in clinical dentistry, we developed the three-dimensional pellet culture system using porcine dental pulp cells. This method results in an endogenous scaffold of collagenous extracellular matrix after treatment with BMP2. When human pulp progenitor/stem cells with hydroxyapatite/tricalcium phosphate as a scaffold were implanted into immunocompromised mice, tubular dentin was formed (Gronthos et al., 2000, 2002). The porcine pulp cells grown in the monolayer culture underwent a characteristic process of dedifferentiation and redifferentiation, marked by a loss of 1(I)collagen on day 10 and by an increase of 1(I)collagen and ALPase activity on day 14 following proliferation and differentiation. The expression of Dspp, the differentiation marker of odontoblasts, was increased later, but not that of enamelysin/MMP20 and Phex. In this study, the dental pulp cells exhibited minimal proliferation in the pellet, as analyzed by total cell number, correlating well with previous studies with other cells (Chiba et al., 1998; Lee et al., 2001). The enhanced expression of enamelysin/MMP20 and Phex on day 21 in pellet cultures, compared with monolayer cultures, suggested that the differentiation of pulp cells into odontoblastic lineage was more advanced. Enamelysin/MMP20 is a matrix metalloprotease detected during predentin secretion by odontoblasts (Bègue-Kirn et al., 1998). A phosphatase-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) is an enzyme involved in phosphate homeostasis during odontoblast differentiation (Ruchon et al., 1998). Histological analysis of the pellet, however, has shown that these cells formed osteodentin-like structure but not tubular dentin. In contrast to other studies (Couble et al., 2000; Yokose et al., 2000), differentiation was enhanced without the addition of dexamethasone and organic phosphate/inorganic phosphate. A potential reason for this may be the result of the optimal cell-to-cell interaction and cell-to-matrix interaction, providing a favorable micro-environment/scaffold in pellet cultures.
Next, the effects of rhBMP2 on differentiation were examined. We have previously reported that treatment of monolayer cultures of bovine pulp cells with rhBMP2 significantly increased the expression of Finally, the utility of this pellet culture system treated with BMP2 for dentin regeneration in vivo was investigated. Since the cells in the pellets were surrounded by collagenous matrix, it allowed for convenient manipulation and implantation for cell therapy. Human pulp cells with PGA which were cultured for 24 hrs and implanted into immunocompromised mice expressed BMP2, BMP4, and BMP7 mRNA (Buurma et al., 1999). In vivo protein therapy with BMP2 (Nakashima, 1994a,b) and BMP7 (Rutherford et al., 1993, 1994) and in vivo gene therapy with Bmp11/Gdf11 by ultrasound-mediated gene delivery stimulated reparative dentin formation on the amputated dental pulp (Nakashima et al., 2003). Ex vivo cell therapy may have an advantage, in that the cultured tissue stem/progenitor cells can be implanted after differentiation into odontoblasts and might result in copious amounts of reparative dentin formation. Skin fibroblasts transduced with BMP7-adenovirus induce reparative dentin formation (Rutherford, 2001). The present investigation demonstrated larger amounts of reparative dentin formation on the amputated pulp with a BMP2-supplemented pellet compared with a control pellet. The extracellular matrix of the pellet functions as a natural scaffold, which retains and releases BMPs. Techniques to isolate human pulp stem cells and manipulate their growth under defined in vitro conditions have to be established and optimized before cell therapy with BMP2 can become a clinical reality for caries and endodontic therapy. This investigation is a first step toward that long-term goal of biological regenerative endodontic therapy.
We are grateful to Yamanouchi Pharmaceutical Co., Ltd. for providing recombinant human BMP2. This work was supported by a Grant-in-Aid for Scientific Research (#13470403) from the Ministry of Education, Science, Sports and Culture, Japan. Received for publication January 15, 2004. Revision received June 5, 2004. Accepted for publication June 14, 2004.
Journal of Dental Research, Vol. 83, No. 8,
590-595 (2004) This article has been cited by other articles:
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
