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A novel culture system for porcine odontogenic epithelial cells using a feeder layer
Archives of Oral Biology (2006) 51, 282—290
www.intl.elsevierhealth.com/journals/arob
A novel culture system for porcine odontogenic epithelial cells using a feeder layer M.J. Honda a,*, T. Shimodaira b, T. Ogaeri b, Y. Shinohara a, K. Hata c, M. Ueda a a
Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan b Department of Oral and Maxillofacial Surgery, Nagoya University Postgraduate School of Medicine, Tokyo, Japan c Center for Genetic and Regenerative Medicine, Nagoya University Postgraduate School of Medicine, Tokyo, Japan Accepted 19 September 2005
KEYWORDS 3T3-J2 cells; Ameloblast; Feeder layer; Long-term culture; Odontogenic epithelial cell
Summary The growth of cells in vitro can provide useful models for investigating their behaviour and improving our understanding of their function in vivo. Although the developmental regulation of enamel matrix formation has been comprehensively analysed, the detailed cellular characteristics of ameloblasts remain unclear because of the lack of a system of long-term in vitro culture. Therefore, the establishment of odontogenic epithelial cell lines has taken on a new significance. Here, we report on a novel porcine odontogenic epithelial cell-culture system, which has permitted serial culture of these cells. Epithelial cells were harvested from third molar tooth buds in the fresh mandibles of 6-month-old pigs, and seeded on dishes in D-MEM containing 10% FBS. Before the cells reached confluence, the medium was changed to LHC-9 to select the epithelial cells. When trypsinized epithelial cells were plated together with 3T3-J2 cells as a feeder layer, the epithelial cells grew from single cells into colonies. The colonies then expanded and became confluent, and could be sub-cultured for up to 20 passages. The long-term culture cells expressed mRNA for amelogenin and ameloblastin, as well as enamelysin (MMP-20), which is a tissue-specific gene product unique to ameloblasts. These results show that the system is capable of sustaining the multiplication of odontogenic epithelial cells with the characteristics of ameloblasts. # 2005 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +81 3 5449 5120; fax: +81 3 5449 5121. E-mail address: [email protected] (M.J. Honda). 0003–9969/$ — see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2005.09.005
Odontogenic epithelial cells culture system
Introduction Enamel tissue development is regulated by reciprocal interactions between epithelial ameloblasts and mesenchymal odontoblasts.1 Although odontogenic epithelial cells differentiate into various cell types during enamel formation, the mechanisms responsible for this differentiation are not well understood as, although the culture of a variety of connective tissue types has been achieved, the culture of odontogenic epithelial cells has not. In vitro experiments are crucial for understanding the mechanism of odontogenic epithelial cell differentiation. The establishment of ameloblast primary-culture systems2—5 and ameloblast-like cell lines has been previously reported.6,7 The approach used involved the transformation and immortalization of epithelial cells with the transforming genes of simian virus 40 (SV40).8,9 To date, it has proved difficult to maintain primary odontogenic epithelial cells in cultures for prolonged periods due to their finite lifespan. Recently, one system in which mouse ameloblasts multiply in culture without transformation has been reported,10 but long-term porcine ameloblast culture system from primary odontogenic epithelium has not yet been achieved. Over the last decade, epithelial cells cultured using 3T3-J2 have been useful for the regeneration of tissues for tissue engineering. Previously published reports describe the establishment of a keratinocyte line derived from a mouse teratoma.11,12 Under the special conditions developed for its cultivation (the presence of 3T3-J2 cells at the correct density), this cell line could be propagated indefinitely and maintained its ability to express differentiated functions over a prolonged period. The experimental approach developed in the present work used 3T3-J2 cells as a feeder layer for the odontogenic epithelial cells. The goal of the study was to establish and characterize a long-term-cultured odontogenic epithelial cell lineage that maintained the primary phenotype of odontogenic epithelial cells. By using 3T3-J2 cells as a feeder layer, we have successfully overcome the limitations of odontogenic epithelial cell culture. The passaged cells express the ameloblast-specific markers amelogenin, ameloblastin and enamelysin, but not enamelin. On the other hand, recently, we have previously reported development of a tissue-engineered tooth.13—15 They recognized the tooth structure including enamel, dentin and pulp. However, there has been no report to attempt to make a tooth using a longterm-cultured cell lineage. The development and characterization of these cultured epithelial cells
283 will be useful in future studies of ameloblast biology and tissue engineering.
Materials and methods Isolation and dissociation of porcine third molar tooth buds Enamel organs were harvested as described previously.4 Third molar tooth buds were removed from the fresh mandibles of 6-month-old pigs. In order to separate enamel organ and pulp tissues, the tooth germs were treated with 1000 units/ml of dispase I (Goudou Syuzei, Tokyo, Japan) for 40 min and then mechanically isolated.16 Enamel organ tissues were minced into 2—3 mm3 pieces, treated with 374 units/ml of collagenase (Wako, Osaka, Japan) in Hanks balanced salt solution (HBSS; Invitrogen, Carlsbad, CA, USA) for 50 min at 37 8C, and gently dissociated by trituration. The released cells were passed through a 70-mm cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA) and pelleted by centrifugation (1500 rpm for 8 min). The pellet was resuspended, and 1.0 106 isolated cells were seeded in D-MEM medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO), 100 units/ml penicillin/streptomycin and 2.5 units /ml fungizon. The medium was changed every other day and the cells were cultured under 10% CO2 in air. These cells formed a mixed population of epithelial- and fibroblast-like cells. Before the cells reached confluence, the medium was replaced with LHC-9 (Biofluids, Bethesda, MD, USA) without fetal bovine serum. LHC-9 medium is selective for epithelial cells4 and is based on the MCDB135 medium used by Kukita and co-workers.3 After substitution of LHC-9, the fibroblasts died and were lost from the culture, leaving only the epithelial cells (primary cells).
Preparation of 3T3-J2 cells as feeder layers 3T3-J2 cells (1 104 cell/cm2; a gift from Dr. H. Green, Harvard Medical School) were used as a feeder layer. They were treated with 4 mg/ml mitomycin C (Kyowa Hakko Corp., Tokyo, Japan) in DMEM without serum for 2 h to suppress their proliferation, and washed three times in PBS (GIBCO) to remove the mitomycin C. They were then cultured for 24 h in D-MEM. The treated 3T3-J2 cells acted as a feeder layer and also inhibited the growth of any contaminating fibroblasts.
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Long-term epithelial cell culture The epithelial cells of the primary culture were trypsinized with 0.125% trypsin/0.02% EDTA for 5 min at 37 8C and inoculated over a 3T3-J2 feeder layer (1 105 cell/cm2; first passaged cells). Complete-MEM medium based on a-MEM medium (GIBCO) supplemented with 5% FBS, insulin (5.0 mg/ml), transferrin (5 mg/ml), triiodothyronine (2 1010 M), cholera toxin (1 1010 M), hydrocortisone (0.5 mg/ml), epidermal growth factor (0.1 mg/ml), penicillin (1000 unit/ml), streptomycin (1 mg/ml) and amphotericin B (2.5 mg/ml) was used for the long-term culture. All cultures were maintained at 37 8C in a humidified atmosphere of 10% CO2—air and fed every 2—3 days by replacing the medium with fresh complete-MEM medium. After culturing for 14 days, the epithelial cells were confluent, and were detached by trypsin and passaged. By the time the epithelial cell had reached confluence, the 3T3-J2 cells had disappeared. The cultures were examined using phasecontrast microscopy during 20 passages over 6 months.
Analysis of cell growth We measured the rate of proliferation of the odontogenic epithelial cells using the protocol provided with the WST-8 kit (Wako, Osaka, Japan). To count the epithelial cells, the dishes were first treated with PBS containing 0.2% versene (GIBCO) in order to eliminate the 3T3-J2 cells. The epithelial cells in 12well plates (BD Bioscience, San Jose, CA, USA) were rinsed gently with phosphate-buffered saline (PBS) and an aliquot (200 ml) of 5 mmol [2-(2methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium,monosodium salt] (MTT) was added to the dish followed by incubation for 1 h. The absorbance at 450 nm was read with a spectrophotometer (SmartSpeckTM 3000; BIO-RAD, Tokyo, Japan).
Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total cellular RNA was purified with the TRIZOL reagent (Invitrogen) from porcine third molar tooth buds and samples taken from several passages of the odontogenic epithelial cells. Third molars were used as a positive control, as this developmental stage is known to express amelogenin, ameloblastin, enamelysin and enamelin. For RT-PCR, total RNA was isolated and reverse transcribed with specific primers (SuperscriptTM First-Strand Synthesis System for RT-PCR; Invitrogen). Specific primers were designed based
M.J. Honda et al. on porcine and mouse mRNA sequences. Amelogenin cDNA was amplified with the upstream primer (50 TACGAACCCATGGGTGGA-30 ) and the downstream primer (50 -GAGAACATCGGAGGCAGA-30 ), Ameloblastin cDNA was amplified with the upstream primer (50 ATTCCCAACCTGGCAAGAGG-30 ) and the downstream primer (50 -AGCGCTTTTAATGCCTTTGC-30 ). Enamelysin cDNA was amplified using the upstream primer (50 ATGACTCCTGCAGAAGTGGACA-30 ) and the downstream primer (50 -AGGTCCGTACAGTGCCTGGA-30 ), taken from the sequence of porcine enamelysin. Enamelin cDNA was amplified using the upstream primer (50 -TGAGGAGATGATGCGCTATG-30 ) and the downstream primer (50 -TGAGGTGTCTGGGTTTCCTC-30 ). DSPP,13 BSP and ALP cDNA were also amplified using the upstream primer (DSPP 50 -CAGCCGCTGATTAATATTCCTAAA-3, BSP 50 -ACTGAAGCCCAAGGAACCAC-3, ALP 50 -TCGACCACAGGGTAGGTTTC-3) and the downstream primer (DSPP 50 -TAACATGGGACGTGCAGAAGAACT-30 , BSP50 -GTCCAGAAGACCACGTTG A30 , ALP 50 -CCC TGC AGT TAG GAC TGA GC-30 ) and the housekeeping gene b-actin were amplified the upstream primer (50 -GCGGGGCTACAGCTTCACCAC30 ) and the downstream primer (50 -ATCTCCTTCTGCATCCTGTCG-30 ). PCR was performed with a Platinum PCR Supermix (Invitrogen) at 94 8C for 1 min, followed by 28 cycles at 98 8C for 10 s, at 56 8C (amelogenin, ameloblastin, enamelysin, enamelin and b-actin)/48 8C (BSP)/54 8C (DSPP)/45 8C (ALP), for 1 min and at 72 8C for 1 min. RT-PCR products were visualized by electrophoresis through a 2% agarose gel.
Preparation of biodegradable polymer scaffolds Three-dimensional scaffolds were prepared as described previously17 with the following modifications: polyglycolic acid fiber mesh (PGA scaffold) (fiber diameter = 13 mm; density = 60 mg/ml; Albany International Research Co., Mansfield, MA, USA) was packed into 96-well plates and sanitized in 75% ethanol. The scaffold dimensions were approximately 1 cm3. Before seeding the cells, the scaffolds were collagen-coated overnight at 4 8C (1 mg/ml type I collagen in 10 mM HCl), followed by washing three times in phosphate-buffered saline (PBS) and three times in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA).
In vivo implantation and harvest of cellpolymer constructs The following experiments were conducted in accordance with the Guidelines for Animal Experi-
Odontogenic epithelial cells culture system mentation of the Institute of Medical Science of The University of Tokyo, Japan. Ten male athymic rats (F344/N Jcl-rnu, Chubu Kagaku Shizai, Nagoya, Japan) 4—5-week-old were used in this study. The cultured odontogenic epithelial cell population (1.0 107 cells) was seeded onto PGA scaffolds (n = 10). The animals were anaesthetized with an intraperitoneal injection of sodium pentobarbital (15 mg/kg), and odontogenic epithelial cell-polymer constructs were implanted in the rat omentum.18 Samples developed for 15 weeks were dissected and immediately fixed in freshly prepared 4% (v/v) paraformaldehyde in PBS at 4 8C for 6—8 h. After fixation, the tissues were demineralised for 4 h in 0.2N HCl and, after extensive washing in PBS, they were dehydrated in an ethanol gradient, cleared in xylene and embedded in paraffin. Tissue sections 6-mm thick were mounted on glass slides.
285 The sections were stained with hematoxylin and eosin (H—E)19 or alcian blue (pH 1.0) to detect the presence of sulphated glycosaminoglycans (SGAGs).20
Results Establishment of the long-term odontogenic epithelial cell culture When cells from dissociated whole enamel organ tissue of the porcine third mandibular molar were cultured in D-MEM medium containing 10% FBS and antibiotics, they grew to confluence. The predominant cell type in these cultures had a fibroblastic morphology (Fig. 1a) and proliferated faster than the odontogenic epithelial cells, reaching confluence by day 7. Primary cultures of the odontogenic
Figure 1 Establishment of the odontogenic epithelial cell line: (a) mixed culture of odontogenic epithelial cells and papilla cells. Phase-contrast micrographs of the cells after 1 week of culture. The epithelial cells (e) have formed colonies among the strongly proliferating pulp cells (p); scale bar = 50 mm. (b) Odontogenic epithelial cells in LHC-9 medium. After replacing the D-MEM medium with LHC-9, large monolayer aggregates of epithelial colonies have been formed. The cultured odontogenic epithelial cells are cuboidal or polygonal in appearance (arrowhead); scale bar = 50 mm. (c) Secondary sub-cultured odontogenic epithelial cells on the top of the layer of 3T3-J2 cells after 14 days of cultivation. Cell numbers have increased to form pavement-like monolayers and aggregates of spherical cells. Most cells are cuboidal or polygonal (arrowhead) in appearance; scale bar = 50 mm. (d) Polygonal cells can be seen on top of the 3T3 layer after 14 days of the 10th passage. The odontogenic epithelium cells had the same characteristic morphology throughout the 10 passages; scale bar = 50 mm.
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epithelial cells appeared to contain few contaminating fibroblasts, because the dental follicular and papilla cells were connected by an epithelium that was thought to derive from epithelial cells. When serum was withdrawn from the cells grown in D-MEM and the medium was replaced with LHC9, only polygonal-shaped cells survived and formed small scattered clusters. However, they did not form cellular nodules in this medium, rather they made close cell-to-cell contact with limited extracellular matrix (ECM; Fig. 1b). When the primary cells were transferred to LHC-9 they grew slowly, and many of the non-epithelial cells died and were lost. These findings were consistent with those reported previously.4 Thereafter, the epithelial cells grew successfully for 20 passages when sub-cultured with the 3T3-J2 feeder layer. During subculture, we examined cell morphology using phase-contrast microscopy. No significant changes in morphological appearance were noted over the first 10 passages (Fig. 1b—d) or subsequently (data not shown).
Cell growth The growth curves measured using the MTT assay are shown in Fig. 2a—c. We confirmed that the MTT assay gave readings that were directly proportional to the numbers of cells (data not shown). Fig. 2a compares the proliferation rates of the primary cells between the 1st and 10th passages. Proliferation in the first passage was slower than that from the third passage onwards. At each passage, the number of cells did not increase for the first 12 days and then rapidly increased (Fig. 2a). By the fourth passage at the latest, the rate of proliferation of the odontogenic epithelial cell was constant. The complete-MEM medium used in this study has been widely employed to culture various types of epithelial cell.21,22 To confirm the usefulness of this medium for the growth of odontogenic cells, we compared the multiplication of third-passage cells in complete-MEM medium with that in D-MEM medium plus 10% FBS. The cells grew more rapidly in the complete-MEM medium (Fig. 2b). To determine whether the 3T3-J2 feeder cells facilitated the growth of the odontogenic cells, we sub-cultured first passage and fourth passage cells with and without 3T3-J2 feeder cells. Total cell number in both groups increased from days 1 to 7. There is an increase of first passage cell proliferation level of both with or without 3T3-J2 feeder cells, which is not significant, but becomes significant in case of fourth passage cells. Cell proliferation of fourth passage cells under 3T3-J2 cells was
Figure 2 Cell growth levels were determined at the indicated time points. The data show that the MTT was directly proportional to the number of cells. (a) Comparison of the growth of the odontogenic epithelial cell line in the first 10 passages. Proliferation of the primary cells was slower than that of the subsequent cultures. (b) Comparison of the growth of third-passage cells in complete-MEM medium and in D-MEM with 10% FBS. Cell numbers were more than four-fold greater after 20 days of culture in complete-MEM than in D-MEM medium plus 10% FBS. (c) Growth of first and fourth passage epithelial cells with or without 3T3-J2 cells. Growth of the first passage cells was fairly similar with or without 3T3-J2 cells. However, growth of the fourth passage cells was considerably faster with 3T3-J2 cells than without them.
significantly higher than that without 3T3-J2 cells. It was approximately three-fold higher in the co-cultured cells with 3T3-J2 cells than that in those without 3T3-J2 cells (Fig. 2c).
Odontogenic epithelial cells culture system
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Figure 3 RT-PCR analysis: (a) RNA from isolated porcine tooth buds was analysed using RT-PCR for the expression of epithelial cell-specific markers. Lane 1, amelogenin; lane 2, ameloblastin; lane 3, enamelysin; lane 4, enamelin; lane 5, b-actin. (b) Expression of amelogenin from the 2nd to 10th passage was analysed using RT-PCR. The expected 310-bp amelogenin product was generated. Lane 1, no template, negative control; lanes 2—10, 2nd—10th passage cells; lane 11, 3T3-J2 cells. (c) Expression of ameloblastin from the 2nd to 10th passage was analysed using RT-PCR. The expected 380-bp ameloblastin product was generated. Lanes 1—9, 2nd—10th passage cells; lane 10, 3T3-J2 cells. (d) Expression of enamelysin from the 2nd to 10th passage was analysed using RT-PCR. The expected 400-bp enamelysin product was generated. Lanes 1—9, 2nd—10th passage cells; lane 10, 3T3-J2 cells. (e) Expression of enamelin from the 2nd to 10th passage was analysed using RT-PCR. The expected 315-bp enamelin product was not generated. Lanes 1—9, 2nd—10th passage cells. (f) RT-PCR of b-actin mRNA. Lanes 1—9, 2nd—10th passage cells. Lane 10, 3T3-J2 cells. The 392-bp b-actin product, which serves as a control, was evenly expressed.
Analysis of the gene-expression pattern in the long-term odontogenic epithelial cell cultures We used RT-PCR to examine the expression of a set of ameloblast-specific markers in the epithelial cell cultures. Porcine third-molar enamel organ tissue expressed mRNA for the ameloblast-specific genes amelogenin, ameloblastin, enamelysin and enamelin (Fig. 3a). By contrast, the odontogenic epithelial cells from the 2nd to 10th passages expressed amelogenin (Fig. 3b), ameloblastin (Fig. 3c) and enamelysin (Fig. 3d). Thus, the pattern of expression of amelogenin, ameloblastin and enamelysin during co-culture with 3T3-J2 mimicked the expression pattern of native odontogenic epithelial cells, with the exception of the expression of enamelin (Fig. 3e). On the other hand, the markers for odontoblasts and stratum intermedium, DSPP, BSP and ALP were not expressed in the secondary passaged and eighth passaged odontogenic epithelial cells (data not shown).
Transplantation of the dental epithelial cell line to the omentum of nude rats To test the tissue-forming activity of odontogenic epithelial cells, we transplanted cultured third-pas-
sage cells seeded on PGA scaffolds into the athymic nude rat omentum for 6 weeks. Histological analysis showed that 3 of the 10 grafts retrieved, none produced the new tissue as well as PGA scaffold. Instead, clusters of elongated polarized cells that were similar in appearance to epithelial cells were present in 6 of the 10 grafts (Fig. 4a). By contrast, two of the grafts gave rise to ectopic cartilage tissue (Fig. 4b—d). The ECM around the polygonal cells in the specimen stained with alcian blue (Fig. 4d), suggesting an abundant accumulation of sulphated glycosaminoglycan in the ECM formed by the sub-cultured odontogenic epithelial cells. These results are similar to those reported previously.10 Cluster formation was not observed in control animals receiving only PGA scaffolds and 3T3-J2 cells seeded on the PGA scaffolds (data not shown).
Discussion Various tissue-specific cell-culture systems have been established using 3T3-J2 feeder cells.11,21—25 This approach, which prevents the potential loss of the more highly differentiated cells, was used previously by Green and Rheinwald to establish human epithelial cell lines11,12 and was adopted by us to
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Figure 4 Tissue regenerated in vivo from third passage odontogenic epithelial cells: (a) histology of grafted third passage odontogenic epithelial cells 6 weeks post-implantation displaying epithelial cell clusters within a PGA scaffold (H—E staining); scale bar = 50 mm. (b) Typical hyaline cartilage formed by third passage cells (H—E staining); scale bar = 200 mm. (c) Higher magnification of panel b, showing typical chondrocytes; scale bar = 100 mm. (d) The area shown in panel c stained with alcian blue. The ECM around the cells is stained; scale bar = 100 mm.
overcome the limited lifespan of the odontogenic epithelial cells. Our results can be summarized as follows. First, in our system, the epithelial cells were initially selected in LHC-9 medium from a heterogeneous cell population including mesenchymal cells.3,4 The selected epithelial cells were combined with 3T3-J2 cells as a feeder layer and were able to multiply through numerous passages. The sub-cultured odontogenic epithelial cells within the co-cultures expressed epithelial markers. Moreover, the growth rate of the sub-cultured epithelial cells in combination with 3T3-J2 cells was substantially greater than without the feeder cells, although the mechanism of this proliferative effect was not established in this study. Second, after 10 passages the cell morphology remained similar to that of the primary cultured cells. Third, we investigated the changes in expression of ameloblast-specific mRNA during long-term culture. No obvious changes were detected in amelogenin, ameloblastin, enamelysin and b-actin gene expression, although interestingly, enamelin was not expressed during the 10 passages. As amelogenin, ameloblastin and enamelysin are expressed in both immature and mature amelo-
blasts, whereas enamelin is expressed only in mature ameloblasts,26—28 it is possible that the ameloblasts remained immature in our culture system. One may postulate that the feeder 3T3-J2 cells, which facilitated growth for the ameloblasts also prevented enamelin production. Finally, recently it is well known that the odontoblasts and stratum intermedium were expressed the mRNA for odontogenic epithelial cell markers.29,30 Therefore, we investigated whether the long-term epithelial cell cultures were expressed both the odontoblasts and stratum intermedium markers, and these multiple cells were not expressed in their markers. The growth of the cells of the fourth passage was markedly different from that of the first passage cells. The fourth passage cells finally stopped growing in the absence of 3T3-J2 cells and could not be sub-cultured in a further fifth passages. After fourth passage the cells, feeder cells is required to maintain the phenotype of the primary porcine ameloblast. After several passaged, it might be the progenitor populations under the no feeder layer may be induce the terminal differentiation and lost
Odontogenic epithelial cells culture system the capacity of the growth. It seems that the first passage cells had the capacity to proliferate without 3T3-J2 cells, and that the phenotype of the first passaged cells was similar with that of the primary cell. To estimate the tissue-forming activity of the cultured odontogenic epithelial cells, we transplanted them on PGA scaffolds into the athymic nude rat omentum and observed ectopic cartilage formation. The regenerated cartilage appeared to be composed of mature chondrocytes, and no bone structures or tooth-like tissues were detected. Two critical points can be made from the data presented above. First, it is already reported that the specific low molecular mass amelogenin gene splice products have the ability to enhance the transcription factor Sox 9.31,32 Therefore, one may postulate that the secreted amelogenin from implanted odontogenic epithelial cells induce the chondrocyte from the immature mesenchymal cells. Second, our result was similar to a previous report describing cartilage regeneration.10 However, in the previous study, bone regeneration was observed when cells of the mouse ameloblast cell lineage were transplanted and they also described that the cultured ameloblast-lineage cells have a different function for inducing either calcification or chondrogenesis.10,33 The difference between the previous report10 and our findings could be due to differences in the effects of growth factors on the odontogenic epithelial cells, because we used a PGA polymer whereas the work with mouse ameloblasts employed Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) that contained several growth factors. Furthermore, it appears that the different amelogenin splice products have different functions.32 Although only limited data were obtained from the present study, we speculate that our established odontogenic epithelial cell line has the capacity for chondrogenesis not osteogenesis. However, it remains to determine why their cells may have formed cartilage and thus future work should focus on whether ameloblasts are a potential source of cells for regenerating cartilage in the field of tissue engineering. Porcine studies are important for regenerative medicine, as several research groups have tried to establish transgenic pigs for xenotransplantation in humans.34—36 For example, Miyagawa et al.37 demonstrated down-regulation of the xenoantigen of pig heart grafts by GnT-III in a pig-to-monkey transplantation model, suggesting that this approach might be useful in clinical xenotransplantation. The factors controlling ameloblast differentiation and their function in enamel formation are not well understood because of two major limita-
289 tions: the limited lifespan of primary cells and the paucity of differentiation markers. We believe that the data presented here are pertinent to this problem. Our findings imply that the multiplied odontogenic epithelial cells using feeder layer are ameloblasts or ameloblast progenitors. Further comprehensive studies will be necessary to clarify the sequence of differentiation events, and the cell lineage of enamel tissue development and tooth tissue regeneration. Recently, many experiments were reported that stem cells exist in the dental mesenchyme.38,39 If these stem cells can be obtained from the dental mesenchyme and be combined with the cultured dental epithelial cell lineage, it could be possible to make a tissueengineered tooth.
Acknowledgements The authors are grateful to Dr. Howard Green at Harvard University for kindly providing 3T3-J2 cells. This work was supported by Grants-in-Aid for Scientific Research, KAKENHI (B) (16390578 to M.H.) and HOUGA (16659548 to M.H.), from the Japan Society for the Promotion of Science, Japan.
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290 10. Nakata A, Kameda T, Nagai H, Ikegami K, Duan Y, Terada K, et al. Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line. Biochem Biophys Res Commun 2003;308:834—9. 11. Rheinwald JG, Green H. Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell 1975;6:317—30. 12. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331—43. 13. Young CS, Terada S, Vacanti JP, Honda M, Bartlett JD, Yelick PC. Tissue engineering of complex tooth structures on biodegradable polymer scaffolds. J Dent Res 2002;81:695—700. 14. Honda MJ, Sumita Y, Kagami H, Ueda M. Histological and immunohistochemical studies of tissue engineered odontogenesis. Arch Histol Cytolo 2005;68:89—101. 15. Honda MJ, Ohara T, Sumita Y, Ogaeri T, Kagami H, Ueda M. Preliminary Study of Tissue-Engineered Odontogenesis in the Canine Jaw. J Oral Maxillofac Surg, in press. 16. Osman M, Ruch JV. Reconstitution of the basement membrane from the inner and outer dental epithelia of trypsinisolated mouse molar enamel organs. J Biol Buccale 1981;9:129—39. 17. Mikos AG, Bao Y, Cima LG, Ingber DE, Vacanti JP, Langer R. Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. J Biomed Mater Res 1993;27:183—9. 18. Choi RS, Vacanti JP. Preliminary studies of tissue-engineered intestine using isolated epithelial organoid units on tubular synthetic biodegradable scaffolds. Transplant Proc 1997;29: 848—51. 19. Stevens A, Lowe L, Bancroft JD. Theory and practice of histological techniques. New York: Churchill Livingstone; 1990. 20. Honda MJ, Yada T, Ueda M, Kimata K. Cartilage formation by serial passaged cultured chondrocytes in a new scaffold:hybrid 75:25 poly(L-lactide-epsilon-caprolactone) sponge. J Oral Maxillofac Surg 2004;62:1510—6. 21. Hata K, Kagami H, Ueda M, Torii S, Matsuyama M. The characteristics of cultured mucosal cell sheet as a material for grafting; comparison with cultured epidermal cell sheet. Ann Plast Surg 1995;34:530—8. 22. Horie K, Kagami H, Hiramatsu Y, Hata K, Shigetomi T, Ueda M. Selected salivary-gland cell culture and the effects of isoproterenol, vasoactive intestinal polypeptide and substance P. Arch Oral Biol 1996;41:243—52. 23. Badylak SF, Record R, Lindberg K, Hodde J, Park K. Small intestinal submucosa: a substrate for in vitro cell growth. J Biomater Sci Polym 1998;9:863—78. 24. De Angeli S, Del Pup L, Fandella A, Di Liddo R, Anselmo G, Macchi C, et al. New experimental models for the in vitro reconstitution of human bladder mucosa. Int J Mol Med 2004;14:367—72.
M.J. Honda et al. 25. Sugimachi K, Sosef MN, Baust JM, Fowler A, Tompkins RG, Toner M. Long-term function of cryopreserved rat hepatocytes in a coculture system. Cell Transplant 2004;13:187— 95. 26. Deutsch D, Catalano-Sherman J, Dafni L, David S, Palmon A. Enamel matrix proteins and ameloblast biology. Connect Tissue Res 1995;32:97—107. 27. Diekwisch TG, Ware J, Fincham AG, Zeichner-David M. Immunohistochemical similarities and differences between amelogenin and tuftelin gene products during tooth development. J Histochem Cytochem 1997;45:859—66. 28. Hu JC, Sun X, Zhang C, Simmer JP. A comparison of enamelin and amelogenin expression in developing mouse molars. Eur J Oral Sci 2001;109:125—32. 29. Papagerakis P, MacDougall M, Hotton D, Bailleul-Forestier I, Oboeuf M, Berdal A. Expression of amelogenin in odontoblasts. Bone 2003;32:228—40. 30. Bourd-Boittin K, Septier D, Hall R, Goldberg M, Menashi S. Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor. J Histochem Cytochem 2004;52:437—45. 31. Nebgen DR, Inoue H, Sabsay B, Wei K, Ho CS, Veis A. Identification of the chondrogenic-inducing activity from bovine dentin (bCIA) as a low-molecular-mass amelogenin polypeptide. J Dent Res 1999;78:1484—94. 32. Veis A, Tompkins K, Alvares K, Wei K, Wang L, Wang XS, et al. Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo. J Biol Chem 2000;275:41263—72. 33. Huggins C, Wiseman S, Reddi AH. Transformation of fibroblasts by allogeneic and xenogeneic transplants of demineralized tooth and bone. J Exp Med 1970;132:1250—8. 34. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science 2000;289:1188—90. 35. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002;295:1089—92. 36. Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science 2003;299:411—4. 37. Miyagawa S, Murakami H, Takahagi Y, Nakai R, Yamada M, Murase A, et al. Remodeling of the major pig xenoantigen by N-acetylglucosaminyltransferase III in transgenic pig. J Biol Chem 2001;276:39310—9. 38. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81:531—5. 39. Braut A, Kollar EJ, Mina M. Analysis of the odontogenic and osteogenic potentials of dental pulp in vivo using a Col1a1-2.3-GFP transgene. Int J Dev Biol 2003;47: 281—92.
www.intl.elsevierhealth.com/journals/arob
A novel culture system for porcine odontogenic epithelial cells using a feeder layer M.J. Honda a,*, T. Shimodaira b, T. Ogaeri b, Y. Shinohara a, K. Hata c, M. Ueda a a
Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan b Department of Oral and Maxillofacial Surgery, Nagoya University Postgraduate School of Medicine, Tokyo, Japan c Center for Genetic and Regenerative Medicine, Nagoya University Postgraduate School of Medicine, Tokyo, Japan Accepted 19 September 2005
KEYWORDS 3T3-J2 cells; Ameloblast; Feeder layer; Long-term culture; Odontogenic epithelial cell
Summary The growth of cells in vitro can provide useful models for investigating their behaviour and improving our understanding of their function in vivo. Although the developmental regulation of enamel matrix formation has been comprehensively analysed, the detailed cellular characteristics of ameloblasts remain unclear because of the lack of a system of long-term in vitro culture. Therefore, the establishment of odontogenic epithelial cell lines has taken on a new significance. Here, we report on a novel porcine odontogenic epithelial cell-culture system, which has permitted serial culture of these cells. Epithelial cells were harvested from third molar tooth buds in the fresh mandibles of 6-month-old pigs, and seeded on dishes in D-MEM containing 10% FBS. Before the cells reached confluence, the medium was changed to LHC-9 to select the epithelial cells. When trypsinized epithelial cells were plated together with 3T3-J2 cells as a feeder layer, the epithelial cells grew from single cells into colonies. The colonies then expanded and became confluent, and could be sub-cultured for up to 20 passages. The long-term culture cells expressed mRNA for amelogenin and ameloblastin, as well as enamelysin (MMP-20), which is a tissue-specific gene product unique to ameloblasts. These results show that the system is capable of sustaining the multiplication of odontogenic epithelial cells with the characteristics of ameloblasts. # 2005 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +81 3 5449 5120; fax: +81 3 5449 5121. E-mail address: [email protected] (M.J. Honda). 0003–9969/$ — see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2005.09.005
Odontogenic epithelial cells culture system
Introduction Enamel tissue development is regulated by reciprocal interactions between epithelial ameloblasts and mesenchymal odontoblasts.1 Although odontogenic epithelial cells differentiate into various cell types during enamel formation, the mechanisms responsible for this differentiation are not well understood as, although the culture of a variety of connective tissue types has been achieved, the culture of odontogenic epithelial cells has not. In vitro experiments are crucial for understanding the mechanism of odontogenic epithelial cell differentiation. The establishment of ameloblast primary-culture systems2—5 and ameloblast-like cell lines has been previously reported.6,7 The approach used involved the transformation and immortalization of epithelial cells with the transforming genes of simian virus 40 (SV40).8,9 To date, it has proved difficult to maintain primary odontogenic epithelial cells in cultures for prolonged periods due to their finite lifespan. Recently, one system in which mouse ameloblasts multiply in culture without transformation has been reported,10 but long-term porcine ameloblast culture system from primary odontogenic epithelium has not yet been achieved. Over the last decade, epithelial cells cultured using 3T3-J2 have been useful for the regeneration of tissues for tissue engineering. Previously published reports describe the establishment of a keratinocyte line derived from a mouse teratoma.11,12 Under the special conditions developed for its cultivation (the presence of 3T3-J2 cells at the correct density), this cell line could be propagated indefinitely and maintained its ability to express differentiated functions over a prolonged period. The experimental approach developed in the present work used 3T3-J2 cells as a feeder layer for the odontogenic epithelial cells. The goal of the study was to establish and characterize a long-term-cultured odontogenic epithelial cell lineage that maintained the primary phenotype of odontogenic epithelial cells. By using 3T3-J2 cells as a feeder layer, we have successfully overcome the limitations of odontogenic epithelial cell culture. The passaged cells express the ameloblast-specific markers amelogenin, ameloblastin and enamelysin, but not enamelin. On the other hand, recently, we have previously reported development of a tissue-engineered tooth.13—15 They recognized the tooth structure including enamel, dentin and pulp. However, there has been no report to attempt to make a tooth using a longterm-cultured cell lineage. The development and characterization of these cultured epithelial cells
283 will be useful in future studies of ameloblast biology and tissue engineering.
Materials and methods Isolation and dissociation of porcine third molar tooth buds Enamel organs were harvested as described previously.4 Third molar tooth buds were removed from the fresh mandibles of 6-month-old pigs. In order to separate enamel organ and pulp tissues, the tooth germs were treated with 1000 units/ml of dispase I (Goudou Syuzei, Tokyo, Japan) for 40 min and then mechanically isolated.16 Enamel organ tissues were minced into 2—3 mm3 pieces, treated with 374 units/ml of collagenase (Wako, Osaka, Japan) in Hanks balanced salt solution (HBSS; Invitrogen, Carlsbad, CA, USA) for 50 min at 37 8C, and gently dissociated by trituration. The released cells were passed through a 70-mm cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA) and pelleted by centrifugation (1500 rpm for 8 min). The pellet was resuspended, and 1.0 106 isolated cells were seeded in D-MEM medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO), 100 units/ml penicillin/streptomycin and 2.5 units /ml fungizon. The medium was changed every other day and the cells were cultured under 10% CO2 in air. These cells formed a mixed population of epithelial- and fibroblast-like cells. Before the cells reached confluence, the medium was replaced with LHC-9 (Biofluids, Bethesda, MD, USA) without fetal bovine serum. LHC-9 medium is selective for epithelial cells4 and is based on the MCDB135 medium used by Kukita and co-workers.3 After substitution of LHC-9, the fibroblasts died and were lost from the culture, leaving only the epithelial cells (primary cells).
Preparation of 3T3-J2 cells as feeder layers 3T3-J2 cells (1 104 cell/cm2; a gift from Dr. H. Green, Harvard Medical School) were used as a feeder layer. They were treated with 4 mg/ml mitomycin C (Kyowa Hakko Corp., Tokyo, Japan) in DMEM without serum for 2 h to suppress their proliferation, and washed three times in PBS (GIBCO) to remove the mitomycin C. They were then cultured for 24 h in D-MEM. The treated 3T3-J2 cells acted as a feeder layer and also inhibited the growth of any contaminating fibroblasts.
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Long-term epithelial cell culture The epithelial cells of the primary culture were trypsinized with 0.125% trypsin/0.02% EDTA for 5 min at 37 8C and inoculated over a 3T3-J2 feeder layer (1 105 cell/cm2; first passaged cells). Complete-MEM medium based on a-MEM medium (GIBCO) supplemented with 5% FBS, insulin (5.0 mg/ml), transferrin (5 mg/ml), triiodothyronine (2 1010 M), cholera toxin (1 1010 M), hydrocortisone (0.5 mg/ml), epidermal growth factor (0.1 mg/ml), penicillin (1000 unit/ml), streptomycin (1 mg/ml) and amphotericin B (2.5 mg/ml) was used for the long-term culture. All cultures were maintained at 37 8C in a humidified atmosphere of 10% CO2—air and fed every 2—3 days by replacing the medium with fresh complete-MEM medium. After culturing for 14 days, the epithelial cells were confluent, and were detached by trypsin and passaged. By the time the epithelial cell had reached confluence, the 3T3-J2 cells had disappeared. The cultures were examined using phasecontrast microscopy during 20 passages over 6 months.
Analysis of cell growth We measured the rate of proliferation of the odontogenic epithelial cells using the protocol provided with the WST-8 kit (Wako, Osaka, Japan). To count the epithelial cells, the dishes were first treated with PBS containing 0.2% versene (GIBCO) in order to eliminate the 3T3-J2 cells. The epithelial cells in 12well plates (BD Bioscience, San Jose, CA, USA) were rinsed gently with phosphate-buffered saline (PBS) and an aliquot (200 ml) of 5 mmol [2-(2methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium,monosodium salt] (MTT) was added to the dish followed by incubation for 1 h. The absorbance at 450 nm was read with a spectrophotometer (SmartSpeckTM 3000; BIO-RAD, Tokyo, Japan).
Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total cellular RNA was purified with the TRIZOL reagent (Invitrogen) from porcine third molar tooth buds and samples taken from several passages of the odontogenic epithelial cells. Third molars were used as a positive control, as this developmental stage is known to express amelogenin, ameloblastin, enamelysin and enamelin. For RT-PCR, total RNA was isolated and reverse transcribed with specific primers (SuperscriptTM First-Strand Synthesis System for RT-PCR; Invitrogen). Specific primers were designed based
M.J. Honda et al. on porcine and mouse mRNA sequences. Amelogenin cDNA was amplified with the upstream primer (50 TACGAACCCATGGGTGGA-30 ) and the downstream primer (50 -GAGAACATCGGAGGCAGA-30 ), Ameloblastin cDNA was amplified with the upstream primer (50 ATTCCCAACCTGGCAAGAGG-30 ) and the downstream primer (50 -AGCGCTTTTAATGCCTTTGC-30 ). Enamelysin cDNA was amplified using the upstream primer (50 ATGACTCCTGCAGAAGTGGACA-30 ) and the downstream primer (50 -AGGTCCGTACAGTGCCTGGA-30 ), taken from the sequence of porcine enamelysin. Enamelin cDNA was amplified using the upstream primer (50 -TGAGGAGATGATGCGCTATG-30 ) and the downstream primer (50 -TGAGGTGTCTGGGTTTCCTC-30 ). DSPP,13 BSP and ALP cDNA were also amplified using the upstream primer (DSPP 50 -CAGCCGCTGATTAATATTCCTAAA-3, BSP 50 -ACTGAAGCCCAAGGAACCAC-3, ALP 50 -TCGACCACAGGGTAGGTTTC-3) and the downstream primer (DSPP 50 -TAACATGGGACGTGCAGAAGAACT-30 , BSP50 -GTCCAGAAGACCACGTTG A30 , ALP 50 -CCC TGC AGT TAG GAC TGA GC-30 ) and the housekeeping gene b-actin were amplified the upstream primer (50 -GCGGGGCTACAGCTTCACCAC30 ) and the downstream primer (50 -ATCTCCTTCTGCATCCTGTCG-30 ). PCR was performed with a Platinum PCR Supermix (Invitrogen) at 94 8C for 1 min, followed by 28 cycles at 98 8C for 10 s, at 56 8C (amelogenin, ameloblastin, enamelysin, enamelin and b-actin)/48 8C (BSP)/54 8C (DSPP)/45 8C (ALP), for 1 min and at 72 8C for 1 min. RT-PCR products were visualized by electrophoresis through a 2% agarose gel.
Preparation of biodegradable polymer scaffolds Three-dimensional scaffolds were prepared as described previously17 with the following modifications: polyglycolic acid fiber mesh (PGA scaffold) (fiber diameter = 13 mm; density = 60 mg/ml; Albany International Research Co., Mansfield, MA, USA) was packed into 96-well plates and sanitized in 75% ethanol. The scaffold dimensions were approximately 1 cm3. Before seeding the cells, the scaffolds were collagen-coated overnight at 4 8C (1 mg/ml type I collagen in 10 mM HCl), followed by washing three times in phosphate-buffered saline (PBS) and three times in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA).
In vivo implantation and harvest of cellpolymer constructs The following experiments were conducted in accordance with the Guidelines for Animal Experi-
Odontogenic epithelial cells culture system mentation of the Institute of Medical Science of The University of Tokyo, Japan. Ten male athymic rats (F344/N Jcl-rnu, Chubu Kagaku Shizai, Nagoya, Japan) 4—5-week-old were used in this study. The cultured odontogenic epithelial cell population (1.0 107 cells) was seeded onto PGA scaffolds (n = 10). The animals were anaesthetized with an intraperitoneal injection of sodium pentobarbital (15 mg/kg), and odontogenic epithelial cell-polymer constructs were implanted in the rat omentum.18 Samples developed for 15 weeks were dissected and immediately fixed in freshly prepared 4% (v/v) paraformaldehyde in PBS at 4 8C for 6—8 h. After fixation, the tissues were demineralised for 4 h in 0.2N HCl and, after extensive washing in PBS, they were dehydrated in an ethanol gradient, cleared in xylene and embedded in paraffin. Tissue sections 6-mm thick were mounted on glass slides.
285 The sections were stained with hematoxylin and eosin (H—E)19 or alcian blue (pH 1.0) to detect the presence of sulphated glycosaminoglycans (SGAGs).20
Results Establishment of the long-term odontogenic epithelial cell culture When cells from dissociated whole enamel organ tissue of the porcine third mandibular molar were cultured in D-MEM medium containing 10% FBS and antibiotics, they grew to confluence. The predominant cell type in these cultures had a fibroblastic morphology (Fig. 1a) and proliferated faster than the odontogenic epithelial cells, reaching confluence by day 7. Primary cultures of the odontogenic
Figure 1 Establishment of the odontogenic epithelial cell line: (a) mixed culture of odontogenic epithelial cells and papilla cells. Phase-contrast micrographs of the cells after 1 week of culture. The epithelial cells (e) have formed colonies among the strongly proliferating pulp cells (p); scale bar = 50 mm. (b) Odontogenic epithelial cells in LHC-9 medium. After replacing the D-MEM medium with LHC-9, large monolayer aggregates of epithelial colonies have been formed. The cultured odontogenic epithelial cells are cuboidal or polygonal in appearance (arrowhead); scale bar = 50 mm. (c) Secondary sub-cultured odontogenic epithelial cells on the top of the layer of 3T3-J2 cells after 14 days of cultivation. Cell numbers have increased to form pavement-like monolayers and aggregates of spherical cells. Most cells are cuboidal or polygonal (arrowhead) in appearance; scale bar = 50 mm. (d) Polygonal cells can be seen on top of the 3T3 layer after 14 days of the 10th passage. The odontogenic epithelium cells had the same characteristic morphology throughout the 10 passages; scale bar = 50 mm.
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epithelial cells appeared to contain few contaminating fibroblasts, because the dental follicular and papilla cells were connected by an epithelium that was thought to derive from epithelial cells. When serum was withdrawn from the cells grown in D-MEM and the medium was replaced with LHC9, only polygonal-shaped cells survived and formed small scattered clusters. However, they did not form cellular nodules in this medium, rather they made close cell-to-cell contact with limited extracellular matrix (ECM; Fig. 1b). When the primary cells were transferred to LHC-9 they grew slowly, and many of the non-epithelial cells died and were lost. These findings were consistent with those reported previously.4 Thereafter, the epithelial cells grew successfully for 20 passages when sub-cultured with the 3T3-J2 feeder layer. During subculture, we examined cell morphology using phase-contrast microscopy. No significant changes in morphological appearance were noted over the first 10 passages (Fig. 1b—d) or subsequently (data not shown).
Cell growth The growth curves measured using the MTT assay are shown in Fig. 2a—c. We confirmed that the MTT assay gave readings that were directly proportional to the numbers of cells (data not shown). Fig. 2a compares the proliferation rates of the primary cells between the 1st and 10th passages. Proliferation in the first passage was slower than that from the third passage onwards. At each passage, the number of cells did not increase for the first 12 days and then rapidly increased (Fig. 2a). By the fourth passage at the latest, the rate of proliferation of the odontogenic epithelial cell was constant. The complete-MEM medium used in this study has been widely employed to culture various types of epithelial cell.21,22 To confirm the usefulness of this medium for the growth of odontogenic cells, we compared the multiplication of third-passage cells in complete-MEM medium with that in D-MEM medium plus 10% FBS. The cells grew more rapidly in the complete-MEM medium (Fig. 2b). To determine whether the 3T3-J2 feeder cells facilitated the growth of the odontogenic cells, we sub-cultured first passage and fourth passage cells with and without 3T3-J2 feeder cells. Total cell number in both groups increased from days 1 to 7. There is an increase of first passage cell proliferation level of both with or without 3T3-J2 feeder cells, which is not significant, but becomes significant in case of fourth passage cells. Cell proliferation of fourth passage cells under 3T3-J2 cells was
Figure 2 Cell growth levels were determined at the indicated time points. The data show that the MTT was directly proportional to the number of cells. (a) Comparison of the growth of the odontogenic epithelial cell line in the first 10 passages. Proliferation of the primary cells was slower than that of the subsequent cultures. (b) Comparison of the growth of third-passage cells in complete-MEM medium and in D-MEM with 10% FBS. Cell numbers were more than four-fold greater after 20 days of culture in complete-MEM than in D-MEM medium plus 10% FBS. (c) Growth of first and fourth passage epithelial cells with or without 3T3-J2 cells. Growth of the first passage cells was fairly similar with or without 3T3-J2 cells. However, growth of the fourth passage cells was considerably faster with 3T3-J2 cells than without them.
significantly higher than that without 3T3-J2 cells. It was approximately three-fold higher in the co-cultured cells with 3T3-J2 cells than that in those without 3T3-J2 cells (Fig. 2c).
Odontogenic epithelial cells culture system
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Figure 3 RT-PCR analysis: (a) RNA from isolated porcine tooth buds was analysed using RT-PCR for the expression of epithelial cell-specific markers. Lane 1, amelogenin; lane 2, ameloblastin; lane 3, enamelysin; lane 4, enamelin; lane 5, b-actin. (b) Expression of amelogenin from the 2nd to 10th passage was analysed using RT-PCR. The expected 310-bp amelogenin product was generated. Lane 1, no template, negative control; lanes 2—10, 2nd—10th passage cells; lane 11, 3T3-J2 cells. (c) Expression of ameloblastin from the 2nd to 10th passage was analysed using RT-PCR. The expected 380-bp ameloblastin product was generated. Lanes 1—9, 2nd—10th passage cells; lane 10, 3T3-J2 cells. (d) Expression of enamelysin from the 2nd to 10th passage was analysed using RT-PCR. The expected 400-bp enamelysin product was generated. Lanes 1—9, 2nd—10th passage cells; lane 10, 3T3-J2 cells. (e) Expression of enamelin from the 2nd to 10th passage was analysed using RT-PCR. The expected 315-bp enamelin product was not generated. Lanes 1—9, 2nd—10th passage cells. (f) RT-PCR of b-actin mRNA. Lanes 1—9, 2nd—10th passage cells. Lane 10, 3T3-J2 cells. The 392-bp b-actin product, which serves as a control, was evenly expressed.
Analysis of the gene-expression pattern in the long-term odontogenic epithelial cell cultures We used RT-PCR to examine the expression of a set of ameloblast-specific markers in the epithelial cell cultures. Porcine third-molar enamel organ tissue expressed mRNA for the ameloblast-specific genes amelogenin, ameloblastin, enamelysin and enamelin (Fig. 3a). By contrast, the odontogenic epithelial cells from the 2nd to 10th passages expressed amelogenin (Fig. 3b), ameloblastin (Fig. 3c) and enamelysin (Fig. 3d). Thus, the pattern of expression of amelogenin, ameloblastin and enamelysin during co-culture with 3T3-J2 mimicked the expression pattern of native odontogenic epithelial cells, with the exception of the expression of enamelin (Fig. 3e). On the other hand, the markers for odontoblasts and stratum intermedium, DSPP, BSP and ALP were not expressed in the secondary passaged and eighth passaged odontogenic epithelial cells (data not shown).
Transplantation of the dental epithelial cell line to the omentum of nude rats To test the tissue-forming activity of odontogenic epithelial cells, we transplanted cultured third-pas-
sage cells seeded on PGA scaffolds into the athymic nude rat omentum for 6 weeks. Histological analysis showed that 3 of the 10 grafts retrieved, none produced the new tissue as well as PGA scaffold. Instead, clusters of elongated polarized cells that were similar in appearance to epithelial cells were present in 6 of the 10 grafts (Fig. 4a). By contrast, two of the grafts gave rise to ectopic cartilage tissue (Fig. 4b—d). The ECM around the polygonal cells in the specimen stained with alcian blue (Fig. 4d), suggesting an abundant accumulation of sulphated glycosaminoglycan in the ECM formed by the sub-cultured odontogenic epithelial cells. These results are similar to those reported previously.10 Cluster formation was not observed in control animals receiving only PGA scaffolds and 3T3-J2 cells seeded on the PGA scaffolds (data not shown).
Discussion Various tissue-specific cell-culture systems have been established using 3T3-J2 feeder cells.11,21—25 This approach, which prevents the potential loss of the more highly differentiated cells, was used previously by Green and Rheinwald to establish human epithelial cell lines11,12 and was adopted by us to
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Figure 4 Tissue regenerated in vivo from third passage odontogenic epithelial cells: (a) histology of grafted third passage odontogenic epithelial cells 6 weeks post-implantation displaying epithelial cell clusters within a PGA scaffold (H—E staining); scale bar = 50 mm. (b) Typical hyaline cartilage formed by third passage cells (H—E staining); scale bar = 200 mm. (c) Higher magnification of panel b, showing typical chondrocytes; scale bar = 100 mm. (d) The area shown in panel c stained with alcian blue. The ECM around the cells is stained; scale bar = 100 mm.
overcome the limited lifespan of the odontogenic epithelial cells. Our results can be summarized as follows. First, in our system, the epithelial cells were initially selected in LHC-9 medium from a heterogeneous cell population including mesenchymal cells.3,4 The selected epithelial cells were combined with 3T3-J2 cells as a feeder layer and were able to multiply through numerous passages. The sub-cultured odontogenic epithelial cells within the co-cultures expressed epithelial markers. Moreover, the growth rate of the sub-cultured epithelial cells in combination with 3T3-J2 cells was substantially greater than without the feeder cells, although the mechanism of this proliferative effect was not established in this study. Second, after 10 passages the cell morphology remained similar to that of the primary cultured cells. Third, we investigated the changes in expression of ameloblast-specific mRNA during long-term culture. No obvious changes were detected in amelogenin, ameloblastin, enamelysin and b-actin gene expression, although interestingly, enamelin was not expressed during the 10 passages. As amelogenin, ameloblastin and enamelysin are expressed in both immature and mature amelo-
blasts, whereas enamelin is expressed only in mature ameloblasts,26—28 it is possible that the ameloblasts remained immature in our culture system. One may postulate that the feeder 3T3-J2 cells, which facilitated growth for the ameloblasts also prevented enamelin production. Finally, recently it is well known that the odontoblasts and stratum intermedium were expressed the mRNA for odontogenic epithelial cell markers.29,30 Therefore, we investigated whether the long-term epithelial cell cultures were expressed both the odontoblasts and stratum intermedium markers, and these multiple cells were not expressed in their markers. The growth of the cells of the fourth passage was markedly different from that of the first passage cells. The fourth passage cells finally stopped growing in the absence of 3T3-J2 cells and could not be sub-cultured in a further fifth passages. After fourth passage the cells, feeder cells is required to maintain the phenotype of the primary porcine ameloblast. After several passaged, it might be the progenitor populations under the no feeder layer may be induce the terminal differentiation and lost
Odontogenic epithelial cells culture system the capacity of the growth. It seems that the first passage cells had the capacity to proliferate without 3T3-J2 cells, and that the phenotype of the first passaged cells was similar with that of the primary cell. To estimate the tissue-forming activity of the cultured odontogenic epithelial cells, we transplanted them on PGA scaffolds into the athymic nude rat omentum and observed ectopic cartilage formation. The regenerated cartilage appeared to be composed of mature chondrocytes, and no bone structures or tooth-like tissues were detected. Two critical points can be made from the data presented above. First, it is already reported that the specific low molecular mass amelogenin gene splice products have the ability to enhance the transcription factor Sox 9.31,32 Therefore, one may postulate that the secreted amelogenin from implanted odontogenic epithelial cells induce the chondrocyte from the immature mesenchymal cells. Second, our result was similar to a previous report describing cartilage regeneration.10 However, in the previous study, bone regeneration was observed when cells of the mouse ameloblast cell lineage were transplanted and they also described that the cultured ameloblast-lineage cells have a different function for inducing either calcification or chondrogenesis.10,33 The difference between the previous report10 and our findings could be due to differences in the effects of growth factors on the odontogenic epithelial cells, because we used a PGA polymer whereas the work with mouse ameloblasts employed Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) that contained several growth factors. Furthermore, it appears that the different amelogenin splice products have different functions.32 Although only limited data were obtained from the present study, we speculate that our established odontogenic epithelial cell line has the capacity for chondrogenesis not osteogenesis. However, it remains to determine why their cells may have formed cartilage and thus future work should focus on whether ameloblasts are a potential source of cells for regenerating cartilage in the field of tissue engineering. Porcine studies are important for regenerative medicine, as several research groups have tried to establish transgenic pigs for xenotransplantation in humans.34—36 For example, Miyagawa et al.37 demonstrated down-regulation of the xenoantigen of pig heart grafts by GnT-III in a pig-to-monkey transplantation model, suggesting that this approach might be useful in clinical xenotransplantation. The factors controlling ameloblast differentiation and their function in enamel formation are not well understood because of two major limita-
289 tions: the limited lifespan of primary cells and the paucity of differentiation markers. We believe that the data presented here are pertinent to this problem. Our findings imply that the multiplied odontogenic epithelial cells using feeder layer are ameloblasts or ameloblast progenitors. Further comprehensive studies will be necessary to clarify the sequence of differentiation events, and the cell lineage of enamel tissue development and tooth tissue regeneration. Recently, many experiments were reported that stem cells exist in the dental mesenchyme.38,39 If these stem cells can be obtained from the dental mesenchyme and be combined with the cultured dental epithelial cell lineage, it could be possible to make a tissueengineered tooth.
Acknowledgements The authors are grateful to Dr. Howard Green at Harvard University for kindly providing 3T3-J2 cells. This work was supported by Grants-in-Aid for Scientific Research, KAKENHI (B) (16390578 to M.H.) and HOUGA (16659548 to M.H.), from the Japan Society for the Promotion of Science, Japan.
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