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Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 dental papilla mesenchyma cell line that might be used for studying the mechanisms of dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.
Tooth development involves sequential and reciprocal interactions between dental epithelial and mesenchymal cells, and proceeds through a series of cytodifferentiations in specific spatial-temporal patterns (Linde and Goldberg, 1993). Dentinogenesis is a complex process in which multiple signaling pathways converge to induce dentin formation and is controlled by many growth and transcription factors (Thesleff, 2003). The bone morphogenetic proteins (Bmps) are structurally related to the transforming growth factor beta (TGF-β) superfamily and were originally identified by their capacity to induce ectopic bone formation in rodents (Urist, 1965; Wozney et al., 1988). Members of the Bmp family have diverse biological functions during embryonic development (Hogan, 1996; Wu et al., 2003) including a vital role in osteogenesis (Chen et al., 2004; Rosen, 2009). Among the Bmp family members, Bmp2 has been extensively studied for its various biological functions during chondrogenic and osteogenic differentiation (Reddi, 1997; Ducy and Karsenty, 2000). Also, Bmp2 has been shown to promote dental pulp stem cell commitment to the odontoblast lineage in vitro (Yang et al., 2009) and induces dental pulp cell differentiation and mineralization in vitro and in vivo (Nakashima, 2005; Chen et al., 2008). However, detail understandings of the molecular mechanisms of Bmp2 exerting its effects on tooth development and formation remain elusive in particular during postnatal tooth development as homozygous mutant embryos for Bmp2 show developmental abnormalities and die at embryo day 9.5 (Zhang and Bradley, 1996). Recently, conditional Bmp2 knock out (cBmp2-KO) mice were generated and revealed important roles of Bmp2 in later stages of osteogenesis (Bandyopadhyay et al., 2006) and bone fracture healing (Tsuji et al., 2006) as well as other organ development (Ma et al., 2005; Rivera-Feliciano and Tabin, 2006; Lee et al., 2007; Singh et al., 2008). However, roles of Bmp2 during tooth development and formation have not been completely understood. Unlike bone and other tissues, it is relatively hard to collect enough amounts of dental tissues from a single tooth. Therefore, generation of a floxed Bmp2 dental papilla mesenchymal cell line would be a valuable tool for studying the effects of Bmp2 on dental cell lineages as well as relevant molecular events involved in matrix mineralization and dentin regeneration. Such information will help realize the potential of BMP2 as therapeutic agent and for the rational targeting of specific Bmp2 to the appropriate clinical indication.
In this study, we established an immortalized mouse floxed Bmp2 dental papilla mesenchymal cell line using transduction of simian phenotypic and virus 40 T-antigen (SV40). We further observed these cell growth rates and their genotypic and phenotypic characteristics as compared to primary cells. Finally, we tested whether these immortalized cells were inducible by growth factors.
A conditional allele of the mouse Bmp2 gene was created by introducing Cre recombinase recognition sites (loxP), which were placed upstream and downstream of exon 3 to excise the protein-coding region in exon 3 of the Bmp2 gene (Ma and Martin, 2005). Genotyping of floxed Bmp2 animals was done by polymerase chain reaction (PCR) analyses using floxed Bmp2-specific primers (Table 1). Genomic DNA was isolated from the mouse tails by using DNA purification kit, Wizard® Genomic (Promega, Madison, WI). For Bmp2, the floxed allele was amplified as a 400 bp product. Protocols utilized for mouse experiments were approved by the Animal Care and Use for Research of the University of Texas Health Science Center at San Antonio (UTHSCSA), TX, USA.
The dental papilla mesenchymal cells were isolated from the first molars of 1-day-old floxed Bmp2 mice and washed with phosphate-buffered saline (PBS), digested for 1 h at 37°C in a solution of 3 mg/ml collagenase type I and 4 mg/ml of dispase (Worthington Biochem, Freehold, NJ). The cells were grown with alpha minimum essential medium (α-MEM, Invitrogen, San Diego, CA) containing 10% fetal calf serum plus penicillin (100 U/ml) and streptomycin (100 μg/ml), and cultured at 37°C in a humidified atmosphere of air containing 5% CO2. Medium was refreshed every 2 days and cells were spread after reaching confluence.
Primary cells in passage 3 were infected for 24 h with the supernatant of about 85% confluence culture of recombinant lentivirus packaging 293T cells that produced a lentiviral vector carrying the SV 40 large T antigen gene (Applied Biological Materials, Inc., Richmond, BC). Two days after infection, cells were re-plated at a low density. Several colonies were formed, and well-isolated colonies were removed selectively and re-plated at low densities to obtain the secondary selection. Several single cells which grew were expanded into cell lines and passaged at least 30–50 times over a 5- to 12-month period before further analysis. Of the several secondary cell lines established, line iBmp2-dp (immortalized floxed Bmp2 dental papilla mesenchymal) of passage 50 and primary floxed Bmp2 dental papilla mesenchymal cells of passage 3 were used for the following characterization.
Morphology of iBmp2-dp and primary dental papilla mesenchymal cells was observed by a light inverted microscope. iBmp2-dp and primary cell proliferation was identified by 5-bromo-2′-deoxyuridine (BrdU) incorporation. Cells were transferred into four-well glass slides and incubated with 30 μM BrdU (Sigma–Aldrich, St. Louis, MO) in culture medium for 4 h. The cells were treated with a mouse monoclonal anti-BrdU antibody (1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by a 1:1,000 dilution of the secondary antibodies (goat-anti-mouse) with Alexa Fluo1 ® 488 green (Molecular Probes, Eugene, OR). For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst (Sigma–Aldrich). Images were obtained in a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei.
Simian virus 40 sequences were accessed in GenBank (Accession No. J02400) and specific primers were synthesized (Table 1). Genomic DNA was isolated from iBmp2-dp cells of passage 50 and from primary cells of passage 3 as described above. Two hundred nanograms of DNA was diluted in a 25 μl PCR mix of 1× PCR buffer containing 10 pmol of forward and reverse primers, 1 U Red Taq DNA Polymerase and 2.5 mM dNTPs (Sigma–Aldrich). The reactions were carried out at 95°C for 5 min for 1 cycle and then at 95°C for 30 sec, 55°C for 60 sec, and 72°C for 60° sec for 30 cycles, with a final 10 min extension at 72°C. After extension, 5 μl of PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. For detection of SV40 protein expression, iBmp2-dp and primary cells were seeded on coverslips in a six-well plate and cultured for 48 h in standard α-MEM medium. The coverslips were rinsed with PBS and fixed with cold acetone and methanol (1:1). The cells were blocked with 10% goat serum and incubated with a primary anti-SV40 large T antigen monoclonal antibody (1:100, Santa Cruz Biotechnology, Inc.) for 2 h at 37°C. Then the cells were washed 3× for 5 min with 1× PBS and incubated with the secondary antibody with Alexa Fluo ® 568 red (Molecular Probes) for 1 h at room temperature. For negative control, the primary SV40 antibody was replaced by mouse IgG I (Dakocytomation, Carpinteria, CA). For cell nucleus staining, the cells were treated with Hoechst (Sigma–Aldrich). Images of Alexa Fluo ®568 staining of the SV40 protein were obtained at the Core Optical Imaging Facility at UTHSCSA under the same parameters in a Nikon inverted microscope.
For detection of tooth-related proteins, iBmp2-dp, and primary dental papilla mesenchymal cells were fluorescently immunostained by antibodies directed against mouse Bsp and Dmp1 (gifts from Dr. Larry Fisher, NIDCR), Runx2, Osx (Sp7), Opn, Oc, Dsp, and Col1α1 (Santa Cruz Biotechnology, Inc.) and Dlx3 (Abcam, Cambridge, MA). Negative control of mouse IgG 1 was purchased from Dakocytomation. Immunohistochemical assay was performed with corresponding secondary antibodies with Alexa Fluor 488 green fluorescent labeling (Molecular Probes). Microphotographs were obtained under a Nikon microscope using a Nikon Cool pix 4500 digital camera.
Total RNA was extracted from iBmp2-dp and primary cells by using RNA STAT-60 kit (Tel-Test, Inc., Friendswood, TX), treated with DNase I (Promega), and purified with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). RNA concentration was determined at an optical density of OD260. The RNA was transcribed into cDNA by SuperScript II reverse transcriptase (Invitrogen). Specific primers for the PCR was synthesized in Table 1, and this included ATF4, Bsp, Dlx3, Dmp1, Dsp, Oc, Opn, Osn, Osx, Runx2, Gapdh, and collagen type I. The PCR reaction was first denatured at 95°C for 5 min, and then carried out at 95°C for 60 sec, at 55–60°C for 60 sec, and at 72°C for 60 sec for 35 cycles and with a final 10 min extension at 72°C. Five microliters of PCR products were analyzed by agarose gels with ethidium bromide staining. Corrective DNA was verified by DNA sequencing.
For detection of ALP activity, culture of both primary and immortalized dental papilla mesenchymal cells in calcifying medium (α-MEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml), 50 μg/ml ascorbic acid and 10 mM sodium β-glycerophosphate) at given time periods was fixed with 70% ethanol for 5 min and washed in the buffer (100 mM Tris–HCl, pH 9.5; 100 mM NaCl; 50 mM MgCl2). In situ ALP staining was performed according to the supplier’s instructions (Bio-Rad, Hercules, CA). For mineralization assay, iBmp2-dp and primary floxed Bmp2 cells were plated in a six-well plate at a density of 4 × 105 per well and cultured in calcifying medium at 37°C for 13 days. The cells were fixed in 10% formaldehyde neutral buffer solution and then stained with alizarin red S (Sigma–Aldrich).
Primary dental papilla mesenchymal and iBmp2-dp cells were maintained in calcifying medium with or without 10 ng/ml of recombinant human BMP2 (rhBMP2) induction (R&D Systems, Minneapolis, MN). After rhBMP2 induction, cell differentiation, and mineralization were measured by ALP and alizarin red S staining assays.
All values were represented as the mean ± standard deviation (SD). Statistical significance was determined using the unpaired Student’s t-test with a P-value of <0.05 being statistical significant.
To establish Bmp2 conditional mice, a conditional allele of the mouse Bmp2 gene was created by introducing Cre recombinase site (loxP) into regions between intron 2 and intron 3 of the Bmp2 gene (Ma and Martin, 2005). The floxed Bmp2 mice were confirmed by PCR using specific Bmp2 primers (Fig. 1A and Table 1) and South blot analysis (data not shown). To create immortalization of mouse floxed Bmp2 dental papilla mesenchymal cell lines, primary cells were isolated and infected with SV40 and then selected. The transfected cells were formed after 2- to 3-week selections, and these cells bypassed senescence and grew over 50 population doublings (PDLs) without significant growth retardation. One of the transfected cell lines termed iBmp2-dp was passaged at 50 generations and used for detail characterization. SV40 T-Ag gene was detected in iBmp2-dp cells, but not seen in the primary dental papilla mesenchymal cells by PCR analyses (Fig. 1A). Immunohistochemistry further confirmed that simian virus 40 T-Ag was expressed in all of iBmp2-dp cells, whereas immonostaining was not present in the primary cells (Fig. 1B,C).
Cell morphology was studied using a light inverted microscopy (Fig. 2A). iBmp2-dp cells show a spindle shape similar to that of the primary cells. There were no microscopical findings indicating senescence or neoplastic nature in the iBmp2-dp cells. Proliferation of the primary and iBmp2-dp cells appeared stable. However, the iBmp2-dp cells exhibited a more rapid growth rate than that of the primary cells as revealed by BrdU assay (Fig. 2B).
To confirm that iBmp2-dp cells express tooth-related genes similar to the primary dental papilla mesenchymal cells, we measured expression of various tooth-related genes using RT-PCR assay. Figure 3A shows that the transformed cell line synthesized collagenous and non-collagenous protein genes such as collagen type I, ALP, Bsp, Dmp1, Mepe, Oc, Opn, Osn; in particular, Dspp, an important marker of odontogenesis (Ritchie et al., 1994; D’Souza et al., 1997; Chen et al., 2005). The transformed cells also expressed tooth-related transcription factors, ATF4, Dlx3, Osx, and Runx2 (Fig. 3A).
Immunohistochemistry studies further confirmed that these cells express those proteins (Fig. 3B). These results indicate that iBmp2-dp cells with a long-term culture retain their genotypic and phenotypic characteristics similar to the primary dental papilla mesenchymal cells.
To determine the differentiation and mineralization activities of these primary and iBmp2-dp cells, we examined ALP activity by in situ ALP histochemistry as ALP is a marker of dental mesenchymal cell differentiation (MacDougall et al., 1995; Chen et al., 2005). The positive expression of ALP in both the primary and iBmp2-dp cells was seen after a culture of 7 days in calcifying medium (Fig. 4A,B). Also, mineralized nodules in the primary and immortalized dental papilla mesenchymal cells were observed at a 13-day cell culture by using alizarin red S staining (Fig. 4E,F). Furthermore, when both of the primary and iBmp2-dp cells were treated with rhBMP2 for given time periods, BMP2 was able to enhance these cell differentiation and mineralization compared to that of these cells without BMP2 stimulation (Fig. 4C,D,G,H).
In the present study, we were able to establish an immortalized dental papilla mesenchymal cell line iBmp2-dp from primary mouse floxed Bmp2 dental papilla mesenchymal cells at postnatal day 1 by transduction with SV40 T-Ag, a viral oncogene. The immortalized cells stained SV40 positive and retained high ALP activity and strong mineralization ability. Also, iBmp2-dp cells expressed all of the identification markers of tooth-related genes. These data suggest that the cell line iBmp2-dp is functionally active and displays all of genotypic and phenotypic characteristics similar to the primary dental papilla mesenchymal cells. Moreover, the iBmp2-dp cells were inducible and BMP2 stimulated these cell differentiation and mineralization.
The strategy for this study was to introduce SV40 T-Ag into primary floxed Bmp2 dental papilla mesenchymal cells as SV40 T-Ag has been demonstrated to be an effective agent for immortalization manipulation in a variety of studies (Thonemann and Schmalz, 2000; Galler et al., 2006; Wu et al., 2009). Moreover, numerous studies have shown that the viral oncogene SV40 T-Ag is capable of immortalizing various cell phenotypes including mouse (MacDougall et al., 1995; Hanks et al., 1998; Priam et al., 2005), rat (Hayashi et al., 1993; Nagata et al., 1994), bovine (Thonemann and Schmalz, 2000; Iwata et al., 2007), and human (Panagakos, 1998; Galler et al., 2006; Suguro et al., 2008) pulp tissues. These cell lines have been used well in many applications, including the study of normal and pathological cell differentiation and mineralization, the dental tissue repair and regeneration, toxicity and biocompatibility testing, gene regulation and signaling pathway during tooth development.
We observed several transcription factors known to be necessary for tooth development including Runx2, Osx, Dlx3, and ATF4. Both Runx2 and Osx are important factors for not only osteogenesis and chondrogenesis, but also during tooth development (Ducy et al., 1997; Komori et al., 1997; Mundlos et al., 1997; Otto et al., 1997; Nakashima et al., 2002). Mutations of either Runx2 or Osx gene result in abnormal bone and tooth formation and development (Lee et al., 1997; D’Souza et al., 1999; Quack et al., 1999; Nakashima et al., 2002). Dlx3 is a homeodomain transcriptional factor essential for tooth development. Dlx 3 gene mutations are associated with autosomal dominant genetic disorder called tricho-dento-osseous syndrome (TDO) characterized by abnormalities in hair, tooth, and bone development (Price et al., 1998; Dong et al., 2005; Duverger et al., 2008; Wright et al., 2008). ATF4 is necessary for osteoblast differentiation and skeletal development (Yang et al., 2004; Xiao et al., 2005; Ameri and Harris, 2008). Furthermore, it has been shown that these transcriptional factors are regulated by BMP2 and other growth factors (Park and Morasso, 2002; Javed et al., 2008; Matsubara et al., 2008; Ulsamer et al., 2008; Fei et al., 2010). Therefore, the cell line iBmp2-dp or knock out of Bmp2 gene by Cre recombinase in the iBmp2-dp cells will be a valuable asset for studying signaling pathways of tooth development mediated by BMP2.
The iBmp2-dp cells are capable of synthesizing collagen and non-collagenous proteins. Both of these proteins are essential components of dentin extracellular matrix and involved in dentin nucleation and hydroxyapatite. In particular, DSPP defines an important marker during dentinogenesis (Ritchie et al., 1994; D’Souza et al., 1997; Chen et al., 2005). Mutations of DSPP gene are associated with dentinogenesis imperfecta (DGI) (Xiao et al., 2001; Rajpar et al., 2002; Kim and Simmer, 2007), which is the most common dentin genetic disease (MacDougall et al., 2006; Hart and Hart, 2007). We also observed that these immortalized dental papilla mesenchymal cells are able to differentiate and form mineralized nodules when cultured in calcifying medium as well as retain the genotypic and phenotypic characteristics similar to the primary cells although the shape of the immortalized cells did differ a little bit from the primary ones and the growth pattern of the transfected cells displayed a more rapid proliferation rate. Finally, we observed that the iBmp2-dp cells are inducible and BMP2 is able to enhance these cells into differentiation and mineralization.
In summary, the present study demonstrates that the immortalized floxed Bmp2 dental papilla mesenchymal cells have unique odontogenic potential along with expression of odontoblast-specific markers and the capability of forming mineralized matrix. One of advantages in the establishment of such a stable dental papilla mesenchymal cell is to provide a large consistent source of dental papilla mesenchymal cell products like dentin extracellular matrix suitable for biochemical analysis. Another provides excellent resource suitable for Bmp2 knock out experiments with the addition of Cre recombinase (Gagneten et al., 1997; Matsuda and Cepko, 2007). Thus, the iBmp2-dp cells can be a useful cell model for studying the mechanism of Bmp2 effects on dental papilla mesenchyaml cell proliferation, differentiation, and mineralization as well as potential application of these cells for reparative formation and regeneration of dentin.
Contract grant sponsor: National Institute of Health;
Contract grant number: DE019892.
Contract grant sponsor: San Antonio Foundation.
Contract grant sponsor: Natural Science Foundation of China;
Contract grant number: 30801293.
This work was supported in part by National Institute of Health Grant DE019892 (S.C.), San Antonio Area Foundation (S.C.), and the grant from the Natural Science Foundation of China 30801293 (LA Wu) and 2009-10 3M ESPE Preventative Pediatric Dentistry Postdoctoral Research Fellowship of AAPD (LA Wu).