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In vitro Effects of Enamel Matrix Proteins on Rat Bone Marrow Cells and Gingival Fibroblasts
S. Keila1,
C.E. Nemcovsky2,
O. Moses2,
Z. Artzi2 and
M. Weinreb1,*
1 Departments of Oral Biology and
2 Periodontology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv 69978, Israel;
Correspondence: * corresponding author, weinreb{at}post.tau.ac.il
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ABSTRACT
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Emdogain® (EMD), a formulation of Enamel Matrix Proteins (EMP), is used clinically for periodontal regeneration, where it stimulates cementum formation and promotes gingival healing. In this study, we investigated the in vitro effects of EMD on rat bone marrow stromal cells (BMSC) and gingival fibroblasts (GF). EMD (at 25 µg/mL) increased the osteogenic capacity of bone marrow, as evidenced by ~ three-fold increase in BMSC cell number and ~ two-fold increase in alkaline phosphatase (ALP) activity and mineralized nodule formation. The presence of EMD in the initial stages (first 48 hrs) of the culture was crucial for this effect. In contrast, EMD did not induce osteoblastic differentiation of GF (evidenced by lack of mineralization or ALP activity) but increased up to two-fold both their number and the amount of matrix produced. These in vitro data on BMSC and GF could explain the promotive effect of EMD on bone formation and connective tissue regeneration, respectively.
Key Words: enamel proteins • bone marrow • in vitro • rat • gingival fibroblasts • stromal cells
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INTRODUCTION
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Enamel matrix proteins (EMP) are secreted by ameloblasts and serve as important regulators of enamel mineralization (Fincham et al., 1992; Simmer and Fincham, 1995; Robinson et al., 1998). In addition, they are secreted by epithelial cells during root formation and have profound biological roles during the formation of the periodontal supporting tissues, such as the attachment of cementum to root dentin (Ten Cate, 1996), initiation of cementogenesis (Heritier, 1982), and induction of the differentiation of dental follicle cells to cementoblasts (Hammarström, 1997). EMP are composed mainly of amelogenins, with smaller amounts of other non-amelogenin components such as tuftelin, ameloblastin, enamel proteases, etc. (Zeichner-David, 2001).
Due to their important role in the development of the periodontium, EMP were used successfully in pre-clinical and clinical studies for the regeneration of periodontal tissues (Hammarström et al., 1997; Heijl et al., 1997; Mellonig, 1999; Gestrelius et al., 2000; Sculean et al., 2000; Tonetti et al., 2002).
In search of the mechanism whereby EMP induce periodontal regeneration, investigators in both in vivo and in vitro studies suggested that they can act as multipurpose growth factors that stimulate the proliferation of PDL cells (Gestrelius et al., 1997; Davenport et al., 2003; Okubo et al., 2003), or stimulate matrix synthesis and possibly alkaline phosphatase activity of PDL fibroblasts (Gestrelius et al., 1997; Van der Pauw et al., 2000; Haase and Bartold, 2001). Other studies showed that EMP may affect the proliferation or differentiation of osteoblastic cell lines (Schwartz et al., 2000; Jiang et al., 2001). Furthermore, EMP were shown to induce the osteoblastic differentiation of the pluripotential mesenchymal cell line C2C12 (Ohyama et al., 2002). Thus, EMP affect bone cells in a maturation-dependent manner. However, osteoblasts of trabecular and endocortical bone derive from bone marrow osteoprogenitor cells, and the effect of EMP on these cells was not investigated previously. Also, the effect of EMP on gingival fibroblasts (as opposed to PDL fibroblasts) has not been studied extensively. One study reported that EMP increases the proliferation and possibly alkaline phosphatase (ALP) activity in these cells (Van der Pauw et al., 2000). Thus, the aims of the present study were to test the biological effects of EMP on cell number and mineralized tissue formation of rat bone marrow stromal cells and gingival fibroblasts.
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MATERIALS & METHODS
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Animals and EMP
Six-week-old male Sprague-Dawley rats were used for all experiments. Emdogain® (EMD, porcine enamel matrix derivative, as lyophilized protein) was the EMP preparation used in this study (a generous gift of Biora AB, Malmö, Sweden). It was dissolved in  MEM medium prior to dilution to the final concentration. The Tel-Aviv University School of Medicine committee for animal studies approved all experimental handling of the animals in this study.
Rat Femoral Bone Marrow Stromal Cells (BMSC)
Mineralized nodule formation
Cultures of femoral BMSC were initiated as previously described (Weinreb et al., 1997, 1999). Briefly, bone marrow cells were cultured in six-well-plates (Nunc, Roskilde, Denmark) at a density of 2 x 107 cells/well in a medium containing MEM + 13% fetal calf serum (FCS; all reagents except where noted were from Biological Industries, Beit-Haemek, Israel) + 2 mM glutamine + 100 U/mL penicillin + 100 µg/mL streptomycin + 12.5 U/ml Nystatin + 10 mM β-glycerophosphate (Sigma, St. Louis, MO, USA) + 50 µg/mL ascorbic acid (Merck, Darmstadt, Germany) + 10 nM dexamethasone (Sigma) (referred to as full medium). These culture conditions facilitate the differentiation of BMSC to osteoblasts, which produce mineralized matrix in the form of bone-like colonies (nodules), the number of which is proportionate to the number of stromal cells seeded. Control cultures (no EMD) and cultures to which EMD at 25 µg/mL or 100 µg/mL was added were initiated. After an attachment period of 48 hrs, non-adherent cells were removed by being rinsed with PBS (phosphate-buffered saline), and cultures were maintained in 7% CO2 and 37°C for 21 days with twice-a-week medium changes. Cultures were fixed in a 1:1:1.5 solution of 10% formalin/methanol/water for 3 hrs and were stained with the Von Kossa method for minerals. This method stains mineralized matrix in black and non-mineralized matrix in dark yellow. The number of mineralized nodules per well was determined macroscopically with a magnifying glass over transmitted light. The percent dish surface area covered with black stain was measured with a video-based image analysis system (Nova, R&M Biometrics, Nashville, TN, USA).
Alkaline phosphatase (ALP) activity
We also assessed osteoblastic differentiation by measuring ALP activity in culture (Weinreb et al., 1997, 1999). BMSC were cultured as described above either without EMD (control group) or with EMD (25 µg/mL). On day 10, the cells were washed in PBS and scraped in 10 mM Tris-HCl buffer (pH = 7.6) containing 10 mM MgCl2 and 0.1% Triton X-100. ALP activity was determined colorimetrically with p-nitrophenyl phosphate as a substrate. Subsequently, the protein content was measured according to Bradford with the use of a microplate reader at 405 nm and BSA as standard. Enzyme activity was expressed as Units/mg protein.
Timing of EMD addition
To examine whether EMD must be added at seeding time (i.e., in the presence of non-adherent cells) to produce its stimulatory effect, we initiated control cultures (no EMD), and cultures to which EMD (25 µg/mL) was added either immediately upon seeding (time 0) or after the removal of non-adherent cells (which was done 48 hrs post-seeding). Cultures were maintained in full medium for 21 days, stained with the Von-Kossa stain, and mineralized nodule formation was quantitated as described above.
Number of adherent cells
Cultures of BMSC were initiated without EMD or with EMD at 25 µg/mL. After an attachment period of 48 hrs, cells were washed 4 times with PBS to remove non-adherent cells. The adherent cells were collected with 0.25% trypsin +0.02% EDTA (Biological Industries), counted with a hemocytometer, and expressed as percent of the number of cells seeded.
Rat Gingival Fibroblasts (GF)
Cells
Fibroblastic cells were harvested from surgical buccal gingiva explants. Surface epithelium was removed from the explants, and the connective tissue was washed in MEM + 100 U/mL penicillin + 100 µg/mL streptomycin + 12.5 U/mL Nystatin (basic medium). It was then cut into small pieces (< 1.5 mm), which were placed in 25 cm2 culture flasks and cultured in basic medium to which 13% FCS and 2 mM glutamine were added. Explants were incubated at 37°C in humidified air containing 7% CO2, and the medium was changed twice a week. After 3 wks, the cell monolayer surrounding the tissue explants was confluent, and cells were removed with trypsin/EDTA and were re-seeded in six-well plates (Nunc) in full medium (containing Dex). Both primary and first-passage cells showed a typical fibroblastic shape, and the latter were used for these experiments.
Mineralization
GF cells were plated at 1 x 104 or 2 x 104 cells/well and were cultured for 28 days in the absence of EMD (control cultures) or in the presence of EMD (25 µg/mL or 100 µg/mL). Plates were then rinsed in PBS, fixed, and stained with the Von-Kossa method as described above. The optical density of the stained matrix was quantitated with the same image analysis system after correction for the background levels of empty wells.
Determination of the protein content
In addition to quantitation of the stained matrix of the cultures, the protein content of similar, parallel cultures was measured as described in the ALP method above.
Determination of cell number
We initiated parallel cultures by seeding 1 x 104 GF cells without EMD or with EMD (100 µg/mL). On days 2, 5, and 12, cells were removed by trypsin/EDTA and were counted in a hemocytometer.
ALP activity
GF cells were cultured for 10 days, after which ALP activity was measured as described above.
Data are presented as means ± SE, and the results from control and EMD cultures were compared with non-paired t tests.
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RESULTS
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Bone Marrow Stromal Cells
Incubation of BMSC with EMD at 25 µg/mL for 3 wks yielded significantly more (~ two-fold) mineralized nodules (p < 0.005) compared with control cultures (no EMD) (Figs. 1A , 1B). EMD at 100 µg/mL did not produce any effect. Densitometric analysis indicated that the dish area covered with bone-like material was significantly (> four-fold) greater (p < 0.01) in cultures treated with 25 µg/mL EMD, compared with control cultures or cultures treated with 100 µg/mL EMD (Fig. 1C). Therefore, we concluded that EMD at 25 µg/mL enhanced the osteogenic capacity of bone marrow by increasing the number of BMSC differentiating into osteoblasts. The same result was found in 2 separate experiments, and therefore, only the lower EMD concentration was used in the following experiments. In support of our conclusion that EMD enhances osteoblastic differentiation of BMSC, ALP activity was increased ~ two-fold with EMD at 25 µg/mL (Fig. 2A). The number of mineralized nodules formed when EMD was added immediately upon seeding (Time 0) was significantly greater (2.25-fold vs. control, p < 0.005) than when EMD was added after removal of the non-adherent cells, 48 hrs later (40% increase vs. control, p = NS) (Fig. 2B). This indicated that EMD is required immediately upon bone marrow seeding to increase the number of stromal cells which can be recruited to the osteoblastic lineage. In agreement, the number of surviving adherent BMSC 48 hrs post-seeding in the presence of EMD was significantly larger (~ three-fold) compared with that in control cultures (Fig. 2C). Furthermore, EMD increased the protein content of the cultures measured on day 10 (Fig. 2D), attesting to an increased number of cells and their matrix.
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Figure 1. Nodule formation by BMSC. (A) Representative wells stained with the Von-Kossa method showing more mineralized nodules in cultures treated with Emdogain (EMD) at 25 µg/mL. (B) Quantitation of the number of nodules/well (mean ± SE, N = 6/group). Only EMD at 25 µg/mL increased nodule number. ***P < 0.005 (EMD at 25 µg/mL vs. control or 100 µg/mL). (C) Quantitation of the dish area covered with mineralized nodules (mean ± SE, N = 6/group). Only EMD at 25 µg/mL increased nodule area. ***P < 0.005 (EMD at 25 µg/mL vs. control or 100 µg/mL).
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Figure 2. (A) Alkaline phosphatase (ALP) activity of BMSC is enhanced by Emdogain (EMD) at 25 µg/mL (mean ± SE, N = 6/group). **P < 0.01 (EMD vs. control). (B) Quantitation of the number of nodules/well (mean ± SE, N = 6/group). EMD (25 µg/mL) was added immediately upon seeding (Time 0) or 48 hrs later, after removal of non-adherent cells. Only EMD at time 0 increased nodule formation. ***P < 0.005 (EMD at Time 0 vs. control or EMD at 48 hrs). (C) EMD (25 µg/mL) increases the number of adherent BMSC at 48 hrs (mean ± SE, N = 6/group). ***P < 0.005 (EMD vs. control). (D) EMD (25 µg/mL) increases the protein content of BMSC cultures on day 10 (mean ± SE, N = 6/group). *P < 0.05 (EMD vs. control).
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Gingival Fibroblasts
No mineralization occurred when gingival fibroblasts were cultured in the presence of 25 or 100 µg/mL EMD for a period of 4 wks (Fig. 3A). In agreement, the ALP activity of GF cells with or without EMD was negligible (data not shown). However, EMD increased the amount of matrix produced by GF dose-dependently. Quantitative measurements of the optical density of the stained matrix corroborated this visual observation (Fig. 3B). In agreement with these data, the protein content of the cultures was significantly greater in EMD-treated cultures compared with controls (Fig. 3C), the concentration of 100 µg/mL being more effective than 25 µg/mL in producing this effect, in obvious contrast to our findings with BMSC. Finally, EMD (100 µg/mL) increased the number of GF, determined between 2 and 12 days post-seeding, up to two-fold (Fig. 4).
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Figure 3. GF cultures. (A) Representative wells after Von-Kossa staining showing no mineralization but darker color of the matrix, with a greater effect of Emdogain (EMD) at 100 µg/mL than at 25 µg/mL. (B) Average optical density in arbitrary units of the GF matrix. Since darker color is associated with lower OD values (255 = white, 0 = black), the matrix of GF cells treated with EMD stains more intensely (mean ± SE, N = 6/group). ***P < 0.005 vs. control. (C) The protein content of GF cultures increases with the EMD concentration (mean ± SE, N = 6/group). *P < 0.05, ***P < 0.005 (vs. control).
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Figure 4. Emdogain (EMD, 100 µg/mL) increases GF cell number, determined on days 2, 5, or 12 (mean ± SE, N = 6/group). *P < 0.05; **P < 0.01; ***P < 0.005 (vs. the respective control).
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DISCUSSION
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Bone Marrow Stromal Cells
In this study, EMD enhanced the osteogenic potential of bone marrow (i.e., stimulated nodule formation and ALP activity), primarily by increasing the number of stromal cells undergoing osteoblastic differentiation. Stimulation of osteoprogenitor cells in bone marrow could explain the reported ability of EMD to enhance trabecular bone regeneration in the rat femur (Kawana et al., 2001) and around titanium implants in rats (Shimizu-Ishiura et al., 2002). It may also explain the multitude of clinical observations of periodontal bone regeneration with EMD. These in vitro effects of EMD are similar to those of PGE2, a bone anabolic agent (Weinreb et al., 1997, 1999; Keila et al., 2001). Analysis of our data suggests that EMD, like PGE2, maintains the viability of adherent stromal cells and promotes their osteoblastic differentiation. Thus, EMD may exert a survival and a differentiative signal on BMSC, an effect which fits well with its ascribed role in promoting early stages of in vivo bone formation (Kawana et al., 2001; Shimizu-Ishiura et al., 2002) and its reported ability to prolong the growth of murine calvarial osteoblasts (Jiang et al., 2001).
Gingival Fibroblasts
Despite the presence of osteopromotive fetal calf serum and of dexamethasone, 2 agents that support osteoblastic differentiation of rat BMSC, no mineralization occurred when gingival fibroblasts were cultured in the presence of 25 or 100 µg/mL EMD for a period of 4 wks (Fig. 4A ). In support, ALP activity of these cells was negligible. Thus, EMD is not able to induce osteoblastic differentiation of GF, in contrast to their transduction with BMPs (e.g., Krebsbach et al., 2000). Analysis of these data fits well with the notion that EMD is not osteoinductive (Boyan et al., 2000). However, these data differ from a previous report where EMD slightly induced ALP activity in these cells (Van der Pauw et al., 2000), albeit to a much lesser degree than in PDL fibroblasts. These differences could result from species differences (rat vs. human) or culture conditions. Regarding PDL fibroblasts, Gestrelius et al.(1997) reported that EMD promoted mineral nodule formation of these cells; however, Okubo et al., reported that EMD had no effect on osteoblastic differentiation of PDL cells. Thus, it seems that EMD is not osteoinductive on gingival fibroblasts but that PDL fibroblasts may respond to EMD differently from GF. Our data on increased cell number and production of proteinaceous matrix by fibroblasts after exposure to EMD corroborate those reported in many previous studies (Gestrelius et al., 1997; Haase and Bartold, 2001; Okubo et al., 2003). Increased protein content in vitro can result from increased cell number, increased matrix production per cell, or both. Since EMD increased both GF number and protein content to similar degrees, stimulation of cell number seems to be its major effect. This observation could suggest the possible use of EMD in tissue engineering of gingival and connective tissue grafts for root coverage procedures and could also explain the observations of increased width of keratinized tissue following a coronally positioned flap procedure (Hägewald et al., 2002) and faster healing of gingival wounds (Wennström and Lindhe, 2002) when EMD was applied.
Finally, it is noteworthy that the EMD concentration, which exerted its biological effects in our study, differed between BMSC and GF. Certain in vitro studies found that a higher 100 µg/mL concentration leads to better results (Schwartz et al., 2000; Jiang et al., 2001; Kawase et al., 2001; Ohyama et al., 2002); however, others have reported the lower 25 µg/mL concentration to be better (Gestrelius et al., 1997; van der Pauw et al., 2000).
In conclusion, EMD increased BMSC cell number and promoted their osteoblastic differentiation and mineralized matrix production, while it did not promote osteoblastic differentiation of gingival fibroblasts but rather increased their cell number and the amount of matrix produced.
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ACKNOWLEDGMENTS
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This study was supported by the Preminger and Schauder funds of the Tel-Aviv University Faculty of Medicine. Emdogain was kindly provided by Biora AB, Malmö, Sweden. The authors have no conflict of interest in this study.
Received for publication May 17, 2003.
Revision received November 28, 2003.
Accepted for publication December 1, 2003.
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Journal of Dental Research, Vol. 83, No. 2,
134-138 (2004)
DOI: 10.1177/154405910408300210
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ABSTRACT
INTRODUCTION