In Vitro Evaluation of the Odontogenic Potential of Mouse Undifferentiated Pulp Cells

The knowledge generated through basic science research in the fields of stem cell biology, biomaterials and odontogenesis, coupled with recent advances in clinical research in Endodontics, has come together at a time when tissue engineering-based therapies are being translated to clinical practice in Medicine (1). Dental implants are one method of oral rehabilitation fairly investigated and well accepted in dental practice, but factors such as location, blood supply, quality and quantity of bone tissue may interfere with osseointegration and lead to success or failure of this In Vitro Evaluation of the Odontogenic Potential of Mouse Undifferentiated Pulp Cells


INTRODUCTION
The knowledge generated through basic science research in the fields of stem cell biology, biomaterials and odontogenesis, coupled with recent advances in clinical research in Endodontics, has come together at a time when tissue engineering-based therapies are being translated to clinical practice in Medicine (1).Dental implants are one method of oral rehabilitation fairly investigated and well accepted in dental practice, but factors such as location, blood supply, quality and quantity of bone tissue may interfere with osseointegration and lead to success or failure of this

In Vitro Evaluation of the Odontogenic Potential of Mouse Undifferentiated Pulp Cells
therapy (2).The advancement of stem cell research sparked great interest in cultivating them in the presence of signs of odontogenic induction, scheduling them in differentiation of dental cell lineages and, with the aid of a scaffold/extracellular matrix, form a new tooth (3).
Dental stem cells display multifactorial potential such as high proliferation rate, multi-differentiation ability, easy accessibility, high viability and easiness to be induced to distinct cell lineages (4).When odontoblasts are destroyed by caries or restorative procedures, the replacement of these cells can be performed by dental pulp stem cells, which might differentiate into odontoblasts and form reparative dentin Correspondence: Profa.Dra.Karina Fittipaldi Bombonato Prado, Laboratório de Cultura de Células, Departmento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, USP, Avenida do Café, S/N, 14040-904 Ribeirão Preto, SP, Brasil.Tel: +55-16-3602-4052.Fax: +55-16-3602-4794.e-mail: karina@forp.usp.brISSN 0103-6440 Braz Dent J (2012) 23( 4): 328-336 (5).Established cell lines derived from odontoblast-like cells are very useful for a variety of purposes such as to study the synthesis of specific molecules including proteins and mRNA, and the processes of dentinogenesis.This model system can also be employed to study the effects of a variety of molecules such as hormones, growth factors and cytokines on cellular function, in addition to tests with biomaterials or environmental situations with possible effects on odontogenesis (6).Two murine cell lines were developed by Hanks et al. (6), derived from undifferentiated pulp cells (OD-21) and odontoblasts (MDPC-23).Both were developed as spontaneously immortalized cell lines derived from dental papilla cells of the first molar of fetal mice.OD-21 cells are characterized by expressing high baseline of Vascular endothelial growth factor (VEGF) (7), which is found in dentin matrix and its release during caries progression might contribute to the reparative response of pulp-dentinal complex (8).On the other hand, the absence of expression of some markers of odontoblastic activity, such as dentin phosphophoryn (DPP) and dentin sialoprotein (DSP) has been observed in OD-21 cell culture (6).In spite of that, under appropriate conditions, these cells can exhibit mineralization (9).
The purposes of this work were to evaluate the odontogenic potential of OD-21 undifferentiated pulp cells through chemical stimuli in vitro, and to compare the results with those of MDPC-23 cell line, using the following parameters: cell proliferation, cell viability, activity of alkaline phosphatase (ALP), total protein content, detection and quantification of mineralized matrix, immunolocalization of non-collagen proteins (DMP1, ALP and OPN) and real-time polymerase chain reaction (PCR) of genes ALP, DMP1, OSTERIX and RUNX2.

Total Cell Number and Viability
Cells grown for periods of 3, 7 and 10 days were enzymatically detached using 1.3 mg/mL collagenase and 0.25% trypsin solution (Gibco).Total number of cells/ well was determined after Trypan blue (Sigma-Aldrich, St. Louis, MO, USA) staining using a hemacytometer (Hausser Scientific, Horsham, PA, USA).

Total Protein Content and Alkaline Phosphatase Activity
Total protein content was determined at days 3, 7 and 10.Briefly, proteins were extracted from each well with 0.1% sodium lauryl sulfate (Sigma-Aldrich) for 30 min and mixed 1:1 with Lowry solution (Sigma-Aldrich) for 20 min at RT.The extract was diluted in Folin and Ciocalteau's phenol reagent (Sigma-Aldrich) for 30 min at RT. Absorbance was measured at 680 nm using a spectrophotometer (Cecil CE3021, Cambridge, UK).The total protein content was calculated from a standard curve and expressed as μg/mL, which was then normalized by the number of cells.
Alkaline phosphatase (ALP) activity was assayed in the same lysates used for determining total protein content as the release of thymolphthalein from thymolphthalein monophosphate by using a commercial kit (Labtest Diagnóstica, Lagoa Santa, MG, Brazil).Briefly, 50 μL of thymolphthalein monophosphate were mixed with 0.5 mL of 0.3 M diethanolamine buffer, pH 10.1, and left for 2 min at 37 o C. The solution was then added to 50 μL of the lysates obtained from each well for 10 min at 37 o C. For color development, 2 mL of 0.09 M Na 2 CO 3 and 0.25 M NaOH were added.After 30 min, absorbance was measured using a spectrophotometer (CE3021) at 590 nm and ALP activity was calculated from a standard curve using thymolphthalein to give a range from 0.012 to 0.4 μmol thymolphthalein/h/mL. Data were expressed as ALP activity normalized by the number of cells.

Mineralized Matrix Formation
Mineralized matrix formation was detected at day 14 by Alizarin Red S (Sigma-Aldrich) that stains areas rich in calcium.Attached cells were fixed in 10% formalin for 2 h at room temperature.After fixation, the specimens were dehydrated through a graded series of alcohol and stained with 2% Alizarin Red S, pH 4.2, for 10 min.The calcium content was evaluated using a colorimetric method.Briefly, 280 μL of 10% acetic acid was added to each well stained with Alizarin Red S, and the plate was incubated at RT for 30 min under shaking.This solution was transferred to a microcentrifugue tube and after vortexing for 1 min, the slurry was overlaid with 100 μL of mineral oil (Sigma-Aldrich), heated to exactly 85ºC for 10 min, and transferred to ice for 5 min.The slurry was then centrifuged at 20,000 g for 15 min and 100 μL of supernatant was transferred to a new microcentrifugue tube.Then, 40 μL of 10% ammonium hydroxide was added to neutralize the acid and this solution containing 140 μL was read at 405 nm in 96well format using opaque-walled transparent-bottomed plates (Fisher Scientific, Pittsburgh, PA, USA) on the plate reader μQuant (Biotek, Winooski, VT, USA).Data were expressed as absorbance values.

RNA Extraction and Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Gene expression of ALP, DMP1, OSTERIX and RUNX2 were evaluated by means of RT-PCR after 7 days.TaqMan ® Gene Expression Assays (Applied Biosystems, Warrington, UK, USA) were acquired for each gene (RUNX2 Mm 01208365_m1; OSTERIX Mm 00504574_m1; DMP1 Mm 03003491_m1and ALP Mm 01187117).The total RNA from cells was extracted using the Promega RNA extraction kit (Promega, Madison, WI, USA), according to the manufacturer's instructions.The concentration of RNA was determined by optical density at a wavelength of 260 nm, using the Biomate 3 spectrophotometer (Thermospectronic, Rochester, NY, USA).Complementary DNA (cDNA) was synthesized using 2 μg of RNA through a reverse transcription reaction (M-MLV reverse transcriptase, Promega).RT-PCR was performed in a CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using TaqMan ® Gene Expression Assays.

Statistical Analysis
Data were analyzed by SPSS statistical software, version 17.0 (SPSS, Chicago, IL, USA), using the Kruskal-Wallis test followed by Mann-Whitney U test.Three independent experiments for each assay described above were performed to demonstrate reproducibility of the data.Each individual experiment was performed in quintuplicate (n=5) to allow for determination of averages and standard deviation.The significance level of the data was determined at p≤0.05.

Total Cell Number and Viability
The total cell number showed no significant difference among the experimental groups after three days.On the other hand, after 7 and 10 days, the total cell number was significantly smaller for cultures exposed to OM (p<0.05),whereas MDPC-23 cells showed higher proliferation (p<0.05) when compared with the other two groups (Fig. 1).Additionally, cell viability was similar to all groups after 3 and 10 days; after 7 days, the viability of cells exposed to OM was significantly smaller (p<0.05) (Fig. 2).

Total Protein Content (TPC)
There was a decrease in TPC until ten days regarding all the studied groups.Observing each group individually, there was no statistically significant difference in TPC among the groups after three days.On the other hand, after 7 and 10 days, the TPC was significantly higher for cultures grown in the presence of OM, when compared with the other groups (p<0.01) (Fig. 3).

ALP Activity
All the groups presented an increase in the level of ALP activity until 10 days.After 3 days, OD-21+OM group had higher ALP activity when compared with MDPC-23 cells group (p<0.01).On the other hand, after 7 days, odontoblast-like cells showed the higher level of ALP when compared with the other two groups (p<0.01).The cells exposed to OM after 10 days showed similar levels of ALP activity when compared with MDPC-23 cells, and both were significantly higher when compared  with negative control (p<0.01) (Fig. 4).

Mineralized Matrix Formation
Mineralized nodules were detected in MDPC-23 culture plate, but not at OD-21 group.OD-21 group exposed to OM showed a great amount of nodules, despite being smaller than odontoblast-like cells group.In the same way, calcium content measured by the extraction of Alizarin Red S from mineralized matrix after 14 days demonstrated that OD-21 cell culture exposed to OM contained significantly more calcium than the negative control (OD-21) (p<0.01),despite MDPC-23 showing more calcium compared with the other two groups (p<0.01) (Fig. 5).

Immunolabeling
Cell morphology can be seen in Figure 6, demonstrating that MDPC-23 cells are epithelioid and round in shape with multiple cell membrane processes and organized in groups.On the other hand, OD-21 cells are elongated, as well as distributed throughout the field.OD-21 cells exposed to OM had their morphology and distribution slightly modified, with some cells acquiring a polygonal shape.Immunolocalization after 3 days showed that DMP1 protein was highly expressed in MDPC-23 cells and OD-21 + OM cells, when compared with negative control.ALP expression was higher in MDPC-23, followed by OD-21 + OM group and OD-21 group.OPN expression was high in all groups, especially in MDPC-23 and OD-21 groups.

Real-Time PCR
After 7 days, gene expression of ALP was lower in OD-21 +OM group (Fig. 7A) when compared with the other two (p<0.05),whereas DMP1 expression was significantly higher (p<0.05) in MDPC-23 cell cultures (Fig. 7B).The expression of OSTERIX gene was lower for OD-21 + OM (p<0.05) when compared with the other   groups (Fig. 7C).In the case of RUNX2, its expression was higher for OD-21 group when compared with the other groups, which had no statistical difference between them (p<0.05)(Fig. 7D).

DISCUSSION
In response to mild environmental stimuli (attrition or early caries), pre-existing live odontoblasts upregulate their secretory activity and secrete a tubular reactionary dentin matrix (10).On the other hand, strong noxious stimuli (deep caries or pulp exposure) that lead to destruction of existing odontoblasts is followed by formation of reparative dentin secreted by a new generation of odontoblast-like cells derived from dental pulp (11).Reparative dentinogenesis is thought to be dependent on multiple signaling molecules sequestrated in the dentin matrix (12,13).Thus, potential populations of cells within dental pulp capable of giving rise to the new generation of odontoblast-like cells during reparative dentinogenesis are numerous and include the cell-rich layer of Höhl adjacent to the odontoblasts, undifferentiated mesenchymal cells and fibroblasts (10).
Reports related that dental pulp stem cells (DPSCs) isolated from human teeth have the potential of differentiation into odontoblasts (14), showing the capability of these cells to produce mineralized nodules after odonto/osteogenic induction, as well as positivity for markers of both osteogenic and odontogenic differentiation, including ALP, bone sialoprotein (BSP), and dentin sialoprotein (DSP) (15).The same way, PCR data presented in the literature demonstrate that the Ca(OH) 2 solubilized dentin matrix can induce changes in gene expression pattern in several cell types commonly encountered in pulp, including fibroblasts as well in OD-21 and MDPC-23 cell lines, representative of undifferentiated pulp mesenchymal cells and odontoblast-like cells respectively (12), also used in the present investigation.The present study confirmed the in vitro odontogenic potential of OD-21 undifferentiated pulp cells murine cell line.
It was also observed a decrease in the proliferation rate of OD-21 + OM group, especially after 7 and 10 days of culture.The low proliferation rate found in our study might be correlated to a beginning of cell differentiation, supported by investigations with odontoblastic cells (16), which reported that differentiation is preceded by the reduction of cell growth.Thus, a reciprocal relationship between a decrease in cell proliferation and subsequent induction of functional activity might have occurred.In the present investigation, this stimulation of cell function might be demonstrated by the increase in the total protein content in induced cells, in all periods.
Alkaline phosphatase, an early marker of odonto/ osteogenesis, showed a higher activity in induced OD-21 cells after 10 days when compared with uninduced cells (negative control).Plus, there was no statistically significant difference between induced undifferentiated pulp cells and odontoblast-like cells (MDPC-23).ALP, which has been proposed to stimulate mineralization by supplying phosphate or by splitting away inorganic pyrophosphate, is regarded as an important tool for odontoblast differentiation and dentin biomineralization (17).It was also observed a correlation between the high levels of ALP and an increase in mineralized nodules in OD-21 + OM group, as seen through alizarin red staining and colorimetric detection.
The present immunolocalization study demonstrated that DMP1 was expressed in the initial period of culture in odontoblast-like cells more than in undifferentiated pulp cells.Nevertheless, these cells treated with odontogenic medium showed high DMP1 expression, suggesting that this protein plays a role regulating specific genes controlling odontoblast-like differentiation (18).Those authors additionally showed that DMP1 is expressed in the odontoblast-like cells aligned along the reparative dentin-pulp interface.ALP is a protein associated with osteogenic and odontogenic differentiation, and in the present study this protein can be seen expressed mainly in the odontoblast-like cells and induced groups as early as 3 days, suggesting its role in cell differentiation.Some proteins, even not representing specific markers of odontoblastic differentiation, might be normally expressed during reparative dentinogenesis by functional odontoblasts secreting dentin matrix (19).It is the case of osteopontin (OPN), whose expression at an early developmental stage is an indication of its importance as a component in the formation of mineralized tissues like bone and dentin (20).It was also demonstrated that OPN was highly and similar expressed in perinuclear and cytoplasmic regions of odontoblast-like cells as well as in induced and uninduced OD-21 cell groups.
Other important tool used in this investigation was the evaluation of changes in gene expression that may lead to variations in cell functional activity, which are likely to contribute to the regenerative processes observed during dentin formation (12).Analysis of the three groups showed that the patterns of expression of ALP, DMP1, OSTERIX and RUNX2 were distinct after 7 days of culture.ALP expression was lower in the induced undifferentiated pulp cells when compared with the uninduced group.This may suggest that immature cells may contain more odontoblast progenitors and have greater dentinogenic ability necessary for mineralized tissue maturation (21).In the present investigation, DMP1 was expressed by odontoblast-like cells.The levels of DMP1 were significantly increased at day 7 in MDPC-23 cells when compared with induced and uninduced undifferentiated pulp cells.The low levels of DMP1 in the OD-21 cells at this period are corroborated by other investigations (10), demonstrating that DMP1 gene was not detected at day 7, appeared at day 14 and increased at day 21.These data suggest that its expression occurs at later stages, playing a role in biomineralization, regulation of phosphate homeostasis and cell differentiation (22).OSTERIX had its expression higher in OD-21 and MDPC-23 cell groups when compared with OD-21 + OM, indicating that chemical stimuli used in our experiments did not favor the expression of this gene, which has been shown to be expressed in differentiated odontoblasts and to be associated with cell differentiation and tooth-related gene expression at the later stages of tooth development via a RUNX2-independent signaling pathway (23).
Regarding RUNX2 expression, there were higher levels in the negative control (OD-21) when compared with the other groups.This can be explained by the fact that RUNX2 is essential for tooth development up to the bell stage, being necessary for the formation of the enamel knot, which controls growth and folding of the enamel organ epithelium (24).Other authors corroborate our findings showing that RUNX2 (type II) is strongly expressed in dental papilla mesenchyme, with up-regulation in pre-odontoblasts and no expression in differentiated odontoblasts (25).The results obtained in this investigation suggest that OD-21 undifferentiated pulp cells have potentiality to differentiate into odontoblast-like cells via chemical stimuli, emphasizing the importance of these cells in the process of odontogenesis as well as tissue regeneration.

Figure 1 .
Figure 1.Cell proliferation expressed as number of cells ×10 4 / well after 3, 7 and 10 days of culture.Data are reported as mean ± SD (n=5).

Figure 2 .
Figure 2. Cell viability expressed as percentage of viable cells after 3, 7 and 10 days of culture.Data are reported as mean ± SD (n=5).

Figure 3 .
Figure 3.Total protein content expressed as µg of protein per number of cells at 3, 7 and 10 days of culture.Data are reported as mean ± SD (n=5).

Figure 4 .
Figure 4. Alkaline phosphatase (ALP) activity expressed as μmol thymolphthalein/h per number of cells after 3, 7 and 10 days of culture.Data are reported as mean ± SD (n=5).