Transformation and preliminary characterization of primary human pulp cells

0099-2399/98/2403-0171 $03.00/0 Printed in U.S.A, VOL. 24, No. 3, MARCH1998

JOURNAL OF ENDODONTICS

Copyright © 1998 by The American Association of Endodontists

Transformation and Preliminary Characterization of Primary Human Pulp Cells Fotinos S. Panagakos, DMD, PhD

The odontoblast is the cell responsible for dentin formation and mineralization during tooth development. A number of primary pulp cell culture systems have been used to study the mechanism of dentinogenesis in vitro. One of the difficulties in using primary cells is the limited number of cell divisions they will undergo. In this study, this problem was addressed by transfecting primary cultures of human pulp cells with an SV40-adenovirus construct. This resulted in the establishment of transformed human pulp cells, which were named HPC-T. A series of preliminary experiments were performed to characterize these cells, including their morphology, cell proliferation, alkaline phosphatase production, and cytogenetic make-up. The results demonstrate that SV40-transformed human pulp cells retain many of the characteristics of the parent primary cells and may be useful in the study of pulp cell function in vitro.

(7). Finally, the inability to subculture for multiple passages without senescence can also be an obstacle to long-term studies (8). We have previously isolated human pulp ceils from extracted third molars (9). The pulp tissue was harvested, minced, and the cells obtained by migration from the pulp tissue onto the cell culture dish. These ceils can be passaged only 4 to 5 times before morphological changes become apparent and senescence occurs. To address this problem, other investigators, using different primary human cultures, have attempted to establish and "immortalize" cells by viral transfo~xnation (9-11). In this study, our laboratory reports the transformation and preliminary characterization of primary human pulp cells using an SV40-adenovirus construct.

MATERIALS AND METHODS All cell culture reagents were obtained from Life Technologies (Grand Island, NY). All chemicals and reagents were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. All plasticware was obtained from VWR Scientific (Piscataway, NJ), SV40-adenovirus construct AdSV 1613 was obtained from Dr. K. Jha (Department of Microbiology, New Jersey Medical School, Newark, NJ). The clonal rat pulp cell line RPC-C2A was obtained from Dr. A. Karim (University of Alberta, Alberta, Canada).

Dentinogenesis is a cell-mediated process, regulated by growth factors and cytokines, and modulated by alterations in the extracellular environment. Details of the nature of the cell-mediated events in the process of dentinogenesis in humans have been difficult to define due to the lack of an in vitro human odontoblast cell culture system. In an attempt to address this problem, investigators have developed and used pulp tissue and pulp cell culture systems that can be stimulated to form odontoblast-like cells (1, 2). Investigators have used extracted rodent, bovine, and human teeth as sources of pulp tissue (3-5). The cells can be isolated by either culturing the tissue and obtaining cell outgrowths, or the tissue can be enzymatically digested to allow ~:elease of the cells from the tissue. The isolated pulp cells can then be used to study pulp cell function in vitro. There are a number of difficulties in working with primary cultures of cells. The first is that the cells in the culture are usually heterogeneous (6). This heterogeneity is often difficult to define, whether it is due to the presence of mole than one cell type within the culture or differences in the differentiation state of a single population of cells. The second problem is multiple passages may result in loss of or change in hormone responsiveness of the cells

Isolation of Human Pulp Cells Freshly extracted impacted third molars were obtained from the Department of Oral and Maxillofacial Surgery, New Jersey Dental School, and were immediately placed in phosphate-buffered saline (PBS) supplemented with antibiotics. The crowns and coronal third of the roots were cleaned and any adherent soft tissue removed. The teeth were split open and the pulp tissue removed. The pulp was placed in Hanks' buffered saline solution (HBSS) and washed for I rain. Cultures were established by the migration of puIp ceils from dental pulp explant cultures as previously described (9). All ceils were cultured using Dulbecco's modified Eagle's medium (DMEM), with 10% fetal bovine serum (FBS), and maintained at 37°C in 5% CO2 in a humidified incubator.

Transformation of Primary Pulp Cells Passage 2 primary human pulp cells were plated at a density"of 5 5< 105 cells/75 cma flask and cultured for 24 h. Cells were 171

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washed with PBS and then transfected for 2 h with SV40 construct in serum-free DMEM, at multiplicities of 300 plaque-forming units/cell. Cells were rinsed with PBS and cultured in DMEM and 10% FBS for 24 h. Cells were then trypsinized and plated at a density of 1 X 104 cells/60 mm dish and grown until transformed foci appeared. Cells were passaged and the medium changed every 5 days. The transformed cells were named HPC-T.

Cell Proliferation Cell proliferation was detemained by using a nonradioactive cell proliferation kit (Cell Titer 96, Promega, Madison, WI). Primary human pulp cells and passage 15 (P15) HPC-T cells were detached by trypsin-EDTA treatment and resuspended at a concentration of 1 × 105 cells/ml. Fifty microliters of cell suspension was added to each well of a 96-well cell culture plate. Cells were incubated in DMEM and 10% FBS for 72 h. At the end of the culture period, 15 /xl of tetrazolium dye was added to each well and the culture plate returned to the incubator for an additional 4-h incubation. The dye reaction was terminated by adding 100 /xl solubilization solution to each well. Absorbance was read using a microplate reader at 570 nm (630 nm reference).

Alkaline Phosphatase (AP) Assay Qualitative analysis of AP synthesis was evaluated using histochemical staining as previously described (12). Primary and P15 HPC-T cells (1 X 105 cells/60 mm dish) were plated in DMEM, 10% FBS, 10 nM dexamethasone, and 50 tzg/mt of ascorbic acid and cultured for 6 days. Medium was changed every 48 h. On day 6, plates were fixed and stained using a histochemical AP kit from Sigma. Cells were fixed with 90% (v/v) ethanol, washed with PBS, and incubated for 30 rain at room temperature in a solution containing naphthol-AS-MX phosphate. As a result of the phosphatase activity, the phosphate group is cleaved, and the naphthol-AS-MX is coupled with a diazonium salt, forming an insoluble, visible pigment at the sites of phosphatase activity. Quantitative analysis of AP synthesis was performed as previously described (12). Briefly, primary human pulp cells, P15 HPC-T cells, and RPC-C2A cells were plated at a concentration of 1 X 104 cells/welI into 24-welI ceil culture plates in DMEM, 10% FBS, 10 nM dexamethasone, and 50/zg/ml of ascorbic acid. The culture medium was changed every 48 h. On day 6, cellular fractions from triplicate wells were harvested. The assay system for AP consisted of 0.15 M of 2-amino-2-methyl-1,3-propanediol, 1.0 mM MgC12, and 7.5 mM p-nitrophenylphosphate as substrate and 25/xg of cellular protein. The reaction was conducted for 1 h at 25°C. Absorbance was measured at 405 nm and results compared with a standard curve of AP.

Mineral Staining Primary and P15 HPC-T cells (1 X 105 cells/plate) were cultured in 60 llllTl dishes for 16 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM/3-glycerophosphate, and 50/xg/ml ascorbic acid. Plates were then stained for mineral deposition using a modified yon Kossa staining technique as previously described (12). Fixed cells were incubated at room temperature in 5% silver nitrate for 60 min while exposed to direct light. After incubation,

FIG 1. Light micrograph of preconfluent primary human pulp cells (A) and HPC-T cells (13).Original magnification ×100. plates were rinsed thoroughly with distilled water and then treated with 5% sodium thiosulfate for 2 min.

Chromosome Analysis Chromosome analysis was performed on passage 18 HPC-T cells by the Center for Human and Molecular Genetics, New Jersey Medical School (Newark, NJ).

RESULTS Primary human pulp cells were isolated from pulp tissue obtained from extracted third molars and were transformed using the SV40-adenovirns construct AdSV 1631. Figure 1 is a light micrograph of primary human pulp cells (Fig. 1A) and HPC-T cells (Fig. 1B) at P15. Primary cells display a typical fibroblastic morphology. The culture of HPC-T cells displays a similar morphology; however, a more polygonal-shaped cell apparent versus the culture of primary cells. HPC-T cells proliferate and form confluent monolayers rapidly. Figure 2 depicts proliferation of primary human pulp ceils, HPC-T cells, and the rat clonal pulp cell line RPC-C2A after 72 h in culture. HPC-T cells proliferate at approximately two times the rate of primary pulp cells. RPC-C2A cells had the greatest proliferation rate of all three ceils examined. One observation made with HPC-T cells was that when they achieved confluence, the cells did not form aggregates or multilayered loci, which are commonly seen with transformed cells. Some transformed cells that display this characteristic have also been shown to be tumorigenic. This

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Transformation of Human Pulp Cells

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FIG 2. Proliferation rates of primary human pulp cells, HPC-T cells, and RPC-C2A clonal rat pulp cells. Proliferation rates are presented as a percentage versus primary human pulp cells.

observation of postconfluent HPC-T cells suggests that they may not be tumorigenic. To confirm this observation, however, further studies are necessary. AP is a marker protein of cells that form mineralized matrices (13). We have previously shown that primary human pulp cells are AP-positive (9). In Fig. 3, primary human pulp cells and HPC-T cells were stained for AP after 6 days in culture. A large number

FIG 3. Light micrograph of primary human pulp cells (A) and HPC-T cells stained for AP after 6 days in culture. Original magnification X40.

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FIG 4. AP levels in primary human pulp cells, HPC-T cells, and RPC-C2A cells after 6 days in culture. Cellular fractions were isolated and 25 /~g total protein was assayed for AP. Results are presented in nmol/min/mg of total protein.

of both primary pulp cells (Fig. 3A) and HPC-T cells (Fig. 3B) stain positive for AP. Cellular fractions collected on day 6 postconfluence were analyzed for AP. These results are presented in Fig. 4. Primary cells had 9.88 ± 0.45 units of AP, and HPC-T cells had 10.50 --_ 1.27 units of AP, with 1 unit = 1 nmol/min/mg

FIG 5. Light micrograph of primary human pulp cells (A) and HPC-T cells (B) stained for extracellular matrix mineral deposition after 16 days in culture. Cells were fixed and stained using the von Kossa staining method. Original magnification x40.

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FIG 6. Karyotype of HPC-T cells at passage 18.

protein of AP. For comparison purposes, cellular fractions were also collected form RPC-C2A cells at the same point in culture. These cells had 7.51 + 0.36 units AP, --75% of the AP present in primary pulp cells and HPC-T cells. During extended culture in the presence of ascorbic acid and 13-glycerophosphate, primary human pulp cells establish an extensive extracellular matrix (ECM) that mineralizes. Retention of this capability is critical if HPC-T cells are to be used in future studies involving the secretion and mineralization of a dentin matrix in vitro. In Fgure 5, primary human pulp cells and HPC-T cells were cultured for 16 days and then analyzed for matrix mineralization using the yon Kossa staining method. Primary cells, seen in Fig. 5A, display the formation of mineralized nodules throughout the culture. HPC-T cells, seen in Fig. 5B, display the formation of some mineralized nodules, but these are much smaller and fewer in number when compared with primary human pulp cells. Finally, we performed a chromosomal analysis on HPC-T cells (Fig. 6). Cells at passage 18 were submitted to the Center for Human and Molecular Genetics, New Jersey Medical School (Newark, NJ). The chromosomal number ranged from 56 to 122 per metaphase cell in the 20 cells examined. The median number of chromosomes per cell was 75, and the average was 79.7, Most cells had a hypertriploid chromosomal number. However, morphology of the chromosomes was largely normal.

DISCUSSION Previous research in our laboratory has focused on the regulation of extracellular matrix formation and mineralization by human osteoblasts in vitro (12, 14). Osteoblasts are similar in cellular function to odontoblasts and pulp cells. These studies used a human osteoblastlike cell line, HOS TE85. HOS TE85 cells were originally isolated from an osteosarcoma and subsequently shown to be immortal but nontumorigenic (15). Because this cell line was not used extensively by others in the field of osteoblast cell biology, initial work using these cells focused on characterizing the cell line, determining whether HOS TE85 cells would show typical osteoblastic features, and whether the cells would synthesize an extracellular matrix that calcifies. Many of the techniques used during this process were applied to the work presented herein. In this study, we present a preliminary characterization of primary human pulp cells transformed with an SV40-adenovirus construct. These cells have been designated HPC-T. SV40 has been used previously in the transformation of human cells (10, 11). These cells generally display extended life spans and anchoragedependent growth. HPC-T cells displayed a typical primary pulp cell appearance and demonstrated anchorage-dependent growth. The cells have been passaged once every week (1:5) and have undergone 23 passages thus far with no apparent senescence. The

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cells, when cultured using the same conditions as primary human pulp cells, expressed alkaline phosphatase (AP) and formed a mineralizing ECM within 16 days of plating. HPC-T cells proliferate at --2 times the rate of primary cells. Pulp cell synthesis of AP and proteoglycans has been suggested as a marker of differentiation (16). Decreased pulp cell synthesis of AP has been shown in response to parathyroid hormone (17), a potent stimulator of bone resorption. One of the earliest observations with HPC-T was the lack of uniform, homogeneous AP staining. This observation, combined with our previous experience working with transformed osteoblasts (12, 14), and the presumption that the primary culture that was transformed was also heterogeneous, led to the conclusion that HPC-T is a heterogeneous population of cells. The heterogeneity, reflected in the variations in staining for AP, possibly indicated differences in the differentiation state of the cell line. One of the questions regarding these cells was whether they were now tumorigenic because of the viral transformation, a characteristic seen in other transformed cell lines (18-20). Although it is known that transformation of human cells with SV40 is alone insufficient for cells to become tumorigenic (17), the possibility still existed. It was felt that this heterogeneity, combined with a possible tumorigenic characteristic, would pose difficulty in using these cells for studies on dentinogenesis. One approach in overcoming these potential obstacles is to subculture the HPC-T cells using a limited dilution method and obtaining multiple new clonal lines that could be examined and characterized. The goal of these experiments would be to establish one or more clonal cell lines that are nontumorigenic and function similar to primary cells in vitro. Based on these preliminary characterizations, HPC-T cells seem to be a viable candidate in the process of establishing a clonal human pulp cell line. Further characterization of these cells is necessary. This characterization must include determining whether these cells produce proteins found in the dentin matrix, including collagen type 1, dentin phosphophoryn, and dentin matrix protein- 1, as well as cell responsiveness to many of the growth factors involved in dentin formation. Only after these and other questions are answered can HPC-T cells, or a clonal cell line derived from HPC-T, be presented as a viable alternative to primary pulp cells for use in the study of dentinogenesis. This work was supported in part by the Foundation-University of Medicine and Dentistry of New Jersey. Dr. Panagakos is assistant professor, Department of Prosthodontics and Biomaterials, New Jersey Dental School, Newark, NJ. Address requests for reprints to Dr. Fotinos S. Panagakos, Department of Prosthodontics and Biomaterials, New Jersey Dental School, Newark, NJ 07103.

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