RESEARCH REPORTS Biological
L. Casagrande1,2, F.F. Demarco2,3, Z. Zhang2, F.B. Araujo1, S. Shi4, and J.E. Nör2,5,6*
Dentin-derived BMP-2 and Odontoblast Differentiation
1
Department of Pediatric Dentistry, School of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, Brazil; 2 Angiogenesis Research Laboratory, Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School of Dentistry; 3Department of Operative Dentistry, School of Dentistry, Federal University of Pelotas, Brazil; 4 Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, USA; 5Department of Biomedical Engineering, College of Engineering, University of Michigan; 6Department of Otolaryngology, School of Medicine, University of Michigan, 1011 N. University, Rm. 2309, Ann Arbor, MI 48109-1078, USA; *corresponding author, jenor@umich.edu J Dent Res 89(6):603-608, 2010
Abstract It is known that stem cells from exfoliated deciduous teeth (SHED) can be induced to differentiate into odontoblasts. However, the nature of dentinderived morphogenic signals required for dental pulp stem cell differentiation remains unclear. The hypothesis underlying this work is that dentinderived Bone Morphogenetic Proteins (BMP) are necessary for the differentiation of SHED into odontoblasts. We observed that SHED express markers of odontoblastic differentiation (DSPP, DMP-1, MEPE) when seeded in human tooth slice/scaffolds and cultured in vitro, or implanted subcutaneously into immunodeficient mice. In contrast, SHED cultured in deproteinized tooth slice/scaffolds, or scaffolds without a tooth slice, do not express these markers. SHED express the BMP receptors BMPR-IA, BMPR-IB, and BMPR-II. Notably, blockade of BMP-2 signaling inhibited the expression of markers of odontoblastic differentiation by SHED cultured in tooth slice/ scaffolds. Collectively, this work demonstrates that dentin-derived BMP-2 is required to induce the differentiation of SHED into odontoblasts.
KEY WORDS: endodontics, tissue engineering, stem cells, dental pulp, trauma.
DOI: 10.1177/0022034510364487 Received August 13, 2009; Last revision December 10, 2009; Accepted January 18, 2010 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
Introduction
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tem cells are characterized by their ability to divide to make more stem cells (self-renewal), and to give rise to multiple different types of mature cells (multipotency). These characteristics make a distinction between stem cells and restricted progenitors (e.g., circulating endothelial cell progenitor cells) or differentiated cells, which have a more narrow developmental potential and a reduced ability to proliferate. The human dental pulp has a subpopulation of cells with phenotypic characteristics of stem cells, as demonstrated by their ability to differentiate into a variety of cell types, including neural cells, adipocytes, and odontoblasts (Gronthos et al., 2000; Miura et al., 2003). Recent evidence suggests the possible usefulness of dental pulp stem cells in the repair of bone, cartilage, and the dental pulp tissue itself (for review, see Huang et al., 2009). Stem cells from human exfoliated deciduous teeth (SHED) have been characterized as a stem cell population found in the dental pulp of primary teeth (Miura et al., 2003). More recently, it has been demonstrated that these cells have the potential of regenerating dental pulp in vivo (Cordeiro et al., 2008). Stem cells from primary teeth constitute an attractive autologous source of cells for regenerative endodontics, because trauma leading to necrosis of immature permanent teeth typically happens during the mixed-dentition phase (Andreasen et al., 2007). Several proteins have been used to identify processes related to the differentiation of odontoblasts. Dentin sialophosphoprotein (DSPP) is a highly phosphorylated non-collagenous protein that is cleaved immediately after secretion into 2 daughter proteins, i.e., dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) (Ritchie et al., 1995; MacDougall et al., 1997). DSPP is highly expressed in odontoblasts, although it can also be found in osteoblasts in lower expression levels (Ritchie et al., 1995; MacDougall et al., 1997; Bègue-Kirn et al., 1998; Qin et al., 2002). Dentin Matrix Protein (DMP-1) is expressed by differentiating odontoblasts during development (D’Souza et al., 1997; Toyosawa et al., 2004). The expression of DMP-1 and DSPP in functional odontoblasts in early stages of odontogenesis is consistent with the hypothesis that both DMP-1 and DSPP play a role in the mineralization of dentin (D’Souza et al., 1997; Ye et al., 2004). Matrix extracellular phosphoglycoprotein (MEPE) is a member of the bone matrix protein family
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Materials & Methods Tooth Slice/Scaffolds
Figure 1. Tooth slice/scaffold model and proliferation of SHED in vitro. (A) Highly porous biodegradable PLLA scaffolds were prepared within the pulp chamber of human tooth slices (tooth slice/scaffold devices). (B) As controls, PLLA scaffolds were prepared with the same process as in (A), but without the surrounding tooth slice. (C) We used the WST-1 proliferation assay to evaluate the proliferation of SHED for up to 28 days in vitro. SHED cells were cultured in tooth slice/scaffolds treated with NaOCl for 5 days (for denaturation of dentin proteins), with EDTA for 1 min (to mobilize dentin proteins), or left untreated. As an additional control, SHED cells were cultured in scaffolds without tooth slices. N = 3 wells per experimental condition and time-point. Data are described as mean ± standard deviation, and are representative of 3 independent experiments.
and is involved in the regulation of cellular metabolism during mineralization processes. Analysis of the expression and function of MEPE in differentiating dental pulp stem cells has shown somewhat conflicting results (Liu H et al., 2005; Wei et al., 2007). Notably, none of these markers (individually) can unequivocally demonstrate odontoblastic differentiation. Bone morphogenetic proteins (BMP) were originally identified as protein regulators of cartilage and bone formation and have been involved in embryogenesis and morphogenesis of various organs and tissues, including teeth (Thesleff and Sharpe, 1997; Ike and Urist, 1998). It has become increasingly evident that BMPs play an important role in dentinogenesis and in dentin regeneration (Bessho et al., 1991; Rutherford et al., 1993; Nakashima, 1994; Aberg et al., 1997; Six et al., 2002; Yamashiro et al., 2003). Notably, dentin extracts induce differentiation of dental pulp stem cells and are capable of inducing dentin regeneration (Ike and Urist, 1998; Liu J et al., 2005). However, the role of the dentin-derived BMP in the local differentiation of dental pulp stem cells is not fully understood. The hypothesis underlying this study is that dentin-derived BMP are required to induce differentiation of dental pulp stem cells into odontoblasts.
SHED were cultured in low-glucose Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and used between passages 4 and 6. Extracted non-carious human third molars were collected at the Department of Oral Surgery of the University of Michigan. Tooth slices (1 mm thick) were prepared, pulp tissue was removed, and highly porous Poly-L-lactic acid (PLLA) (molecular weight, 250,000 g/mol; Boehringer Ingelheim, Germany) scaffolds were cast in the pulp cavity, as previously described (Cordeiro et al., 2008). Tooth slice/scaffolds (Fig. 1A) were randomly assigned to the following experimental conditions: (1) 5.25% NaOCl for 5 days at 4°C; (2) 10% EDTA (pH 7.2) for 1 min at 4°C; (3) untreated control tooth slice/scaffolds; and (4) control PLLA scaffolds without the tooth slice (Fig. 1B). SHED (5 x 104) were seeded in the scaffolds and cultured in vitro for 0-28 days. Alternatively, tooth slices seeded with SHED were transplanted subcutaneously into the dorsum of severe combined immunodeficient mice (CB.17 SCID; Charles River, Wilmington, MA, USA), as previously described (Cordeiro et al., 2008). After 14 or 28 days, mice were killed, implants were retrieved, and RNA was extracted for gene expression analysis. The experimental design is summarized in the Appendix Fig. Appropriate institutional review boards at the University of Michigan approved this study.
RT-PCR Analysis SHED cells were cultured for 24 hrs in 0 or 100 ng/mL rhBMP-2 or rhBMP-7 (R&D Systems, Minneapolis, MN, USA). Alternatively, SHED (5 x 104) were seeded into tooth slice/scaffolds and cultured for 0-28 days. In selected experiments, a 2 µg/mL quantity of monoclonal anti-human BMP-2 or BMP-7 antibodies was added to the culture medium (MAB3551, MAB3541; R&D Systems). At the end of the experimental period, RNA was extracted with Trizol (Invitrogen) and reversetranscribed with SuperScript III Platinum (Invitrogen). The following are the primer sequences: GAPDH (sense 5′ gaccccttcattgacctcaact 3′; antisense 5′ caccaccttcttgatgtcatc 3′; 683-bp amplicon); DSPP (sense 5′ gaccccttcattgacctcaact 3′, antisense 5′ tgccatttgctgtgatgttt 3′; 181-bp amplicon); DMP-1 (sense 5′ caggagcacaggaaaaggag 3′, antisense 5′ ctggtggtatcttgggcact 3′; 213-bp amplicon); and MEPE (sense 5′ gcaaaagcacccatcgtatt 3′, antisense 5′ ctgccctctacaaggctgac 3′; 385-bp amplicon). RNA from primary human odontoblasts scraped from freshly extracted teeth was used as positive control. Polymerase chainreaction (PCR) was performed with 35 cycles of denaturation at 94°C for 45 sec, annealing at 57°C for 45 sec, and extension at 72°C for 60 sec. We performed at least 3 independent experiments to verify reproducibility of results.
Western Blots Proteins were retrieved from the following cells: SHED, DPSC, human dental pulp fibroblasts (HDPF), human dermal microvascular endothelial cells (HDMEC), human osteoblast-like cells
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(MG-63), or University of Michigan squamous cell carcinoma cells (UM-SCC)-11A (gift from Tom Carey) in an NP-40-based lysis buffer. Proteins were subjected to SDS gel electrophoresis. Membranes were incubated at 4°C overnight with a 1:500 dilution of the following antibodies: monoclonal anti-human BMPR-IA, BMPR-IB, or BMPR-II antibody (R&D Systems). Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) was used for development of membranes.
WST-1 Proliferation Assay Effects of dentin-derived proteins on SHED proliferation were evaluated in tooth slice/scaffolds seeded with 5 x 104 cells and cultured in vitro for 7-28 days, as described above. At specific time-points, the WST-1 reagent (Roche, Mannheim, Germany) was added to the culture medium at a 1:10 dilution. After onehour incubation (37°C, 5% CO2), supernatants were transferred to 96-well plates, and absorbance was evaluated at 420 nm in a microplate reader (Genius; TECAN, Grödig, Austria).
Results Effects of Dentin Morphogens on SHED Proliferation and Odontoblastic Differentiation To evaluate the effect of dentin on the proliferation of dental pulp stem cells, we cultured, for 28 days, SHED in tooth slice/ scaffolds or in control scaffolds, i.e., scaffolds without tooth slices, or tooth slice/scaffolds that had been previously deproteinized by treatment with 5.25% NaOCl. SHED cultured in scaffolds without dentin showed the highest rate of proliferation among all conditions evaluated (p < 0.05) (Fig. 1C). SHED cultured in tooth slice/scaffolds treated with NaOCl presented higher proliferation rates (p < 0.05) than cells cultured in tooth slice/scaffolds treated with EDTA or left untreated (Fig. 1C). SHED cultured in tissue culture plates did not express the odontoblastic differentiation markers used here, DMP-1, DSPP, and MEPE (Fig. 2A). As positive controls for this experiment, we used primary human odontoblasts, and the negative controls were human dental pulp stem cells (DPSC) and dental pulp fibroblasts (Fig. 2A). To evaluate the effects of dentin morphogens on the differentiation of dental pulp stem cells, we cultured SHED in tooth slice/scaffolds in vitro for up to 28 days. We began to observe expression of markers of odontoblastic differentiation at 14 days in SHED cultured in tooth slice/scaffolds that were either left untreated or treated with EDTA for 1 min (Fig. 2B). In contrast, SHED cultured in tooth slice/scaffolds whose proteins were denatured by exposure to NaOCl for 5 days, or SHED cultured in scaffolds without tooth slices, did not show expression of any of the markers of odontoblastic differentiation utilized here (Fig. 2B). To evaluate whether this effect was simply an artifact of in vitro experimental conditions, we used an identical experimental approach as above, but this time we implanted the tooth slice/ scaffolds subcutaneously into immunodeficient mice (Fig. 2C). We observed that SHED seeded in untreated or EDTA-treated tooth slice/scaffolds, but not in control scaffolds or the NaOCltreated tooth slice/scaffolds, expressed the markers of odontoblastic differentiation (Fig. 2C). Notably, treatment for 1 min with
EDTA potentiated the expression of DMP-1, DSPP, and MEPE in the 28-day time period, as compared with untreated tooth slice/ scaffolds (Fig. 2C). These data correlated well with the histological appearance of the tissues observed within the tooth slice/scaffolds retrieved 28 days after implantation into the mice. We observed that the histological architecture of the tissues generated within EDTA-treated tooth slices resembled more closely the morphology of normal dental pulps, as compared with tissues generated in NaOCl-treated teeth (Fig. 3).
Dentin-derived BMP-2 is Required for the Differentiation of SHED into Odontoblasts Here, we observed that SHED expressed BMPR-IA, BMPR-IB, and BMPR-II (Fig. 4A). SHED exposed to BMP-2 showed strong expression of DSPP, DMP-1, and MEPE, while BMP-7 induced weak expression of DMP-1. To determine the requirement of BMP on dentin-induced odontoblastic differentiation of dental pulp stem cells, we cultured SHED in tooth slice/scaffolds in the presence of neutralizing antibodies to BMP-2 and BMP-7. Blockade of BMP-2 signaling abrogated dentin-induced expression of the 3 markers of odontoblastic differentiation (Fig. 4C). In contrast, blockade of BMP-7 signaling had no effect on the expression of these markers, when compared with the IgG-treated control group (Fig. 4C).
Discussion While much has been learned over the last few years about the ability of dental pulp stem cells to differentiate into multiple cell types, the effects of dentin-derived proteins on the differentiation of dental pulp stem cells into odontoblasts are still largely unknown. Considering that dentin contains bioactive molecules that are capable of stimulating cellular responses important in dentin regeneration (Roberts-Clark and Smith, 2000; Murray and Smith, 2002; Graham et al., 2006), it is critical that we learn which dentin signals are required for odontoblastic differentiation of stem cells. Such knowledge will improve our understanding of the mechanisms involved in dentin regeneration, and will help guide research efforts in dental pulp tissue engineering. Here, we observed that SHED did not express markers of odontoblastic differentiation when they were cultured in scaffolds without dentin. To rigorously evaluate the effects of dentin-derived molecules on the odontoblastic differentiation of SHED, we denatured the dentin proteins with sodium hypochlorite (Beltz et al., 2003). When deproteinized tooth slice/ scaffolds were used, SHED did not express the markers of odontoblastic differentiation. However, SHED seeded in untreated tooth slice/scaffolds were able to express the markers of odontoblastic differentiation. Notably, the results of the SHED proliferation assays provide additional evidence of the role of dentin-derived proteins in odontoblastic differentiation. They demonstrated that, after an initial phase of modest growth between days 0 and 14, the proliferation of SHED seeded in tooth slice/scaffolds is inhibited, which is consistent with the process of terminal differentiation of these cells. In contrast, SHED seeded in NaOCl-treated tooth slice/scaffolds or in control scaffolds (without tooth slices) continued to
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dentin-derived morphogenic signals are necessary to induce odontoblastic differentiation of SHED cells. Our in vitro experiments demonstrated that SHED cultured in untreated and EDTAtreated groups expressed the differentiation markers starting from day 14. In the in vivo experiment, low expression levels of these genes were observed in the 14-day time period, but after 28 days the expression levels of all 3 markers of odontoblastic differentiation were clearly detectable. We hypothesize that the lactic acid released as a by-product of degradation of the PLLA scaffolds “etched” the dentin and facilitated the release of morphogens, which would explain the odontoblastic differFigure 2. Effects of dentin morphogens on the expression of markers of odontoblastic differentiation. (A) entiation of SHED observed in Dental pulp cells (fibroblasts, SHED, and DPSC) grown in regular cell culture plates did not express any the untreated tooth slice/scaffold of the odontoblastic differentiation markers, i.e., DMP-1, DSPP, and MEPE. (B) SHED cells were cultured group. However, the overall in vitro for up to 28 days in tooth slice/scaffolds treated with NaOCl for 5 days (to denature dentin trend observed here was that proteins), with EDTA for 1 min (to mobilize dentin proteins), or left untreated. As an additional control, EDTA-treated teeth presented SHED cells were cultured in scaffolds without tooth slices. Markers of odontoblastic differentiation, i.e., stronger expression of DSPP, DSPP, DMP-1, and MEPE, were evaluated by RT-PCR. (C) SHED cells were seeded in scaffolds prepared as in (B). Tooth slice/scaffolds were implanted subcutaneously into the dorsum of immunodeficient mice. DMP-1, and MEPE when comAfter 14 or 28 days, markers of odontoblastic differentiation (i.e., DSPP, DMP-1, and MEPE) were pared with untreated teeth. It is evaluated by RT-PCR. RNA was pooled from 3 specimens per experimental condition and time-point plausible that EDTA mobilizes (B,C). Data are representative of 3 independent experiments. inducers of odontoblastic differentiation from the dentin (including BMP-2) that enhance expression of markers of differentiation in the SHED. Collectively, these results suggest that, indeed, the mobilization of dentin morphogens by EDTA could be beneficial for regenerative endodontics. Both BMP-2 and BMP-7 have inductive effects in reparative dentinogenesis (Rutherford et al., 1993; Nakashima, 1994; Goldberg et al., 2001). Here, we observed that SHED expressed significant levels of BMP receptors. Therefore, these cells are capable of responding to BMP-2- or BMP-7-mediated signals. When stimulated with recombinant proteins, SHED responded potently to BMP-2 and more modestly to BMP-7. However, Figure 3. Effects of dentin morphogens on the histology of tooth slice/ when we addressed specifically the role of dentin-derived BMP scaffolds containing SHED in vivo (400X). Hematoxylin/eosin staining signaling with neutralizing antibodies, we observed that BMP-2 of representative photomicrographs of tissues generated in the pulp (but not BMP-7) is required for odontoblastic differentiation chamber of tooth slice/scaffolds pre-treated with EDTA (A) or NaOCl under our experimental conditions. Analysis of these data sug6 (B). Tooth slice/scaffolds were seeded with SHED (5 x 10 ) and gests that while BMP-7 has differentiation potential, other sigimplanted subcutaneously into immunodeficient mice for 28 days. D, nals derived from the dentin matrix can effectively substitute for it. dentin; dD, deproteinized dentin (NaOCl-treated); PD, predentin; BV blood vessels; ET, engineered tissue. In contrast, BMP-2 signaling appears to be required for odontoblastic differentiation in the model presented here. These data, however, do not exclude the important role of other inductive proliferate for most of the experimental period. When one conmolecules expressed in the dentin-pulp complex. Studies have siders that continuous cell growth and self-renewal are hallmarks shown unequivocally that transforming growth factor (TGF)of ‘stemness’, analysis of these data suggests that, under condibeta 1 functions on processes that lead to the cytological and tions where dentin proteins are not present, SHED do not diffunctional differentiation of odontoblasts (Bègue-Kirn et al., ferentiate. Analysis of these collective data demonstrated that
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