Pulp cell cultures obtained with two different methods for in vitro cytotoxicity tests O. Cortés*, C. Garcìa*, L. Pérez*, J. Boj**, A. Alcaina* * School of Medicine and Dentistry. University of Murcia. Murcia ** Dental School. University of Barcelona. Barcelona; Spain. Abstract Aim: To describe two different protocols for obtaining primary pulp cell cultures, one derived from explants and the other following dissociation into single cell suspension by enzyme digestion. Methods: Human pulp tissue was obtained from three healthy premolars. The harvested pulp tissue was prepared for culture using physical methods (one of the premolars) and enzyme, type XI collagenase, (the two remaining premolars). Results: In the case of explant based culture, only limited growth was observed in some cases. However, by enzyme digestion, after two weeks cell growth was evident, and differences in cell type were observed according to the tooth involved. Conclusion: It has been possible to obtain abundant biological material using an enzyme digestion-based protocol for testing purposes, with low experimental variability, as all cells originated from the same individual.

Introduction An increasing number of new materials and clinical procedures are introduced in both dental practice and in other fields that require prior evaluation. In paediatric dentistry, despite important advances in the prevention of caries, many teeth are still prematurely lost. Such early dental losses give rise to malocclusion and esthetic, speech and functional problems. One of the main objectives of paediatric dentistry is to preserve the primary dentition until it is replaced by the permanent teeth. In relation to this point, the pulp tissue has important functions to sustain teeth, providing: 1) nutrient supply for dentin/pulp metabolism to maintain the mechanical properties; 2) innervation and sensory organ function for the prevention of deep caries; 3) reparative dentine formation during pulp wound healing process; and 4) immunological response to bacterial infiltration.Nakashima M., 2005. Therefore, all precautions should be taken to preserve the vitality of the primary pulp. In primary teeth when caries are deep and coronal pulp is affected, pulpotomy has been performed. Formocresol has long been the standard pulpotomy medicament in primary teeth, though in recent years its use has been questioned because of the toxic effects. At present, different alternatives are available, including ferric sulphate and mineral trioxide aggregate [Cortés O et al., 1997; Casas et al., 2004; Holan et al., 2005]. Each agent is considered to represent an alternative but requires the prior conduction of pulp biocompati-

bility and toxicity studies. As a result, not only clinical studies but also experimental research involving in vivo and in vitro models is important. Animal research poses a number of problems, as a careful experimental design must first be developed, with adequate selection of the animal species and prior approval by an ethics committee. The animals moreover require adequate care, environmental and housing conditions, and the research personnel must be trained in animal management and these studies sometimes comprise a large number of variables such as marginal microleakage or contamination during the procedures carried out [Langeland et al.1969; Eisenmenger and Zetner,1985; Pascon et al,1991]. On the other hand, in vitro methods offer cell cultures as a complement to animal experimentation and in some disciplines, such as genetics and toxicology, they constitute alternatives to research in animals. Cell culturing comprises those techniques designed to keep plant or animal cells, tissues or organs alive in an adequate nutritional medium. Cell cultures are the most widely employed option, based on already established cell lines or the use of cells freshly harvested from tissues (primary cell cultures). In such cultures the tissues are deliberately disaggregated using physical methods (explants) or enzymes that digest the intercellular bonds to yield independent cells that are transferred to bottles containing a nutritive medium [Jakoby and Pastan, 1979; Freshney,1992; Morgan and Darling,1993]. The main advantages of cell cultures includes the use of human cells for experimentation and control of the environmental conditions. Cell cultures are of use in diagnosing diseases, obtaining drug products, developing assisted reproduction techniques and in evaluating the potential toxic effects of chemical substances destined for human consumption [Morgan and Darling, 1993]. For the biological assessment of such substances, the different international scientific commissions establish cell culture-based cytotoxicity testing as the initially indicated technique Stanford, 1980; International Standards Organization, 1992. In this context, it is possible to investigate pulp tissue biocompatibility based on the assessment of cell viability, with the observation of morphological changes and growth patterns. And, in some cases, functionality, mitogenicity and clonal capacity testing may also be indicated.

Key words: Cell culture, pulp fibroblasts, explants, enzyme digestion, cell viability Postal address: Dr. Olga Cortés, C/ General O’Donnell, 20 3o D,03003 Alicante, Spain Email: [email protected]

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Pulp cell cultures for cytotoxicity tests

Results Most of these studies in our field have been based on the use of established cell lines, due to the difficulty sometimes found in creating primary cultures from harvested tissues, specifically when pulp fibroblasts are involved, as these cells show a low percentage survival [Wen Sun et al., 1990; Hill et al.,1991; Hashieh et al., 1999; Mitchell et al.,1999]. However, the use of primary cultures (in this case pulp fibroblasts) is often desirable, as they may offer increased specific sensitivity to the test agent or substance [van WyK et al., 2001]. The present study describes two different protocols for obtaining primary pulp cell cultures, one derived from explants and the other following dissociation into single cell suspension by enzyme digestion.

Material and methods

The explant-based cultures yielded poor results, as after three weeks only scant cell growth was observed in some of the explants. This method was found to be slower, yielding fewer cells over a longer period of time. However, using enzyme digestion, evident cell growth was initiated after two weeks, with differences between the cultures corresponding to the two original premolars. In effect, in one case (premolar with a fully formed root) fibroblastic cells were identified, with a characteristic fusiform appearance (Figure 1) and orderly and parallel monolayer growth (Figure 2). In the case of the other tooth (incomplete root), differentiated cells were observed exhibiting radial multilayer growth, forming nodules and presenting refringent material at the center of the nodules, similar to osteoblastic cells (Figure 3).

Human pulp tissue from three healthy premolars extracted for orthodontic reasons were used. One tooth presented incomplete root development and corresponded to an 11year-old boy, while the other two teeth had fully formed roots. The three teeth were processed independently. After extraction, and avoiding the risk of contamination, each tooth was immersed in a sterile flask containing Dulbecco (DMEM; Gibco Laboratories, Grand Island, NY, USA) culture medium to which penicillin (100 g/ml), streptomycin (100 g/ml) gentamycin (50 g/ml) and amphotericin (2.5 g/ml) were added (Gibco Laboratories, Grand Island, NY, USA, followed by storage at 4∫C until tissue harvesting. Less than 10 hours later, the pulp tissue was extracted under sterile conditions in a laminar flow chamber. The crowns were sectioned with the help of forceps (with prior creation of a notch along the diameter of the crown at gingival third level). Small amounts of pulp tissue were excavated and placed in a sterile Petri dish.

Figure 1 Microphotograph showing fibroblastic cell, with a fusiform appearance. (Magnification, X20)

One tooth (with a fully formed root) was subjected to tissue disaggregation using physical methods, separating the tissue into small fragments (explants) that were then immersed in a culture flask containing DMEM supplemented with 15% fetal calf serum (FCS; Gibco Laboratories, Grand Island, NY, USA ) to stimulate cell proliferation. The flask was subsequently incubated at 37∫C in and atmosphere containing 7.5% CO2 and with a relative humidity of 90% to prevent culture medium evaporation. In the case of the remaining premolars, and after harvesting the pulp tissue fragments as previously described, the cells were disaggregated using enzyme digestion, in this case type XI collagenase (1.25 mg/ml). After separating the cells by centrifugation (200 g for 10 min.), cell viability was assessed with a haemacytometer based on the trypan blue exclusion test, followed by resuspension in DMEM and incubation. The culture medium was replaced every two days. Phase contrast light microscopic observations were made to assess cell condition and thus determine the timing of subculture once the cultures reached confluence.

Figure 2 Microphotograph showing one month primary pulp cell culture with an orderly and parallel monolayer growth. (Magnification, X20)

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Cortés et al.

Regarding the cell types, most dental studies involving primary cultures use fibroblasts of either pulp origin or obtained from the periodontal ligament. Few studies have used odontoblasts, which require cell phenotypic characterization and sometimes with complex techniques.

Figure 3 Microphotograph showing one month primary pulp cell culture with radial multilayer growth, presenting refringent material at center. (Magnification, X20)

With this enzyme-based method, cell culture was continued for at least four months. During this period subcultures were prepared to obtain cells destined for use in different cytotoxicity tests. The best conditions for subculture comprised treatment with a trypsin/EDTA mixture (0.1/0.2 mg/ml) for only 5 minutes, as longer times induced a decrease in cell viability. The cells were subcultured to 2000 cell/cm2, again reaching confluence after two weeks. After conclusion of the tests the remaining cells were stored in liquid nitrogen (-196∫C) and were defrozen some months later without problems.

Discussion While other studies have reported favorable results with the explants of tissues, [Hill et al., 1991; Keiser et al., 2000] our results with enzyme digestion (type XI collagenase) were superior to those obtained when attempting to mechanically explant small pulp tissue fragments in a culture flask. In this latter case the resulting cell growth was slow and sparse, and was only observed in some of the explanted fragments. However, the number of cells obtained with the primary culture based on enzyme digestion allowed subculturing and the conduction of different tests, and their storage in liquid nitrogen for future studies. In no case did we observe bacterial contamination in primary cultures that may have led to culture failure. Emphasis is therefore placed in these cultures of the importance of the transport conditions, tissue extraction and maintenance of sterility – in addition to the need to apply antibiotherapy to the culture medium. In the present study the combination of four drugs (penicillin, streptomycin, gentamycin and amphotericin) proved adequate, though some authors use only penicillin and streptomycin, while others do not add penicillin [Wen Sun et al.,1990; Keiser et al., 2000; Ashkenazi et al., 2001].

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It should be taken into account that dental pulp constitutes lax connective tissue similar in many aspects to other types of connective tissue but with certain differentiating characteristics, fundamentally its capacity to form calcifications during the tooth formation and development process, and its regenerative and reparatory potential. Fibroblasts are the most abundant type of cell, and are particularly numerous in the cellular zone. These cells seem to persist in a state of relative undifferentiation when compared with the fibroblasts found in most other connective tissue regions, and it is believed that they can differentiate into odontoblasts when necessary. Odontoblasts are the most characteristic cells of the dentinal pulp complex, and their morphology can vary according to the functional activity of the cells [Salentijn and Klyvert, 1985; Davis, 1986]. For this reason it is necessary to confirm the predominant type of cell found in the culture, not only phenotypic characterization based on electron microscopic examination, but also involving other tests such as the assessment of the presence of type I collagen and phosphorin (a protein that participates in dentin mineralization), or of calcified deposits based on the von Kossa stain [Bencosme and Tsutsumi,1970; Nakashima,1991; Kuo, 1992; Tsukamoto et al., 1992]. In the present study it seemed logical to assume that the different cell types observed may be attributable to the different dental root development stages involved. In one case we cultured pulp tissue from a still developing tooth, with a predominance of differentiated cells undergoing active synthesis, while the other tooth had fully formed apexes. In the latter case these cells are less active or are simply in the resting state, and pulp fibroblasts constitute the cell population with the greatest proliferation. We agree with Tsukamoto et al., [1992 that the calcified nodules observed in the former case may correspond to hydroxyapatite. Nevertheless, and in addition to the previously commented on confirmatory tests, it should also be investigated whether the cells are odontoblastic in origin or are differentiated from fibroblasts, taking into account the reparatory capacity of the pulp of the young tooth. On the other hand, the use of primary cultures affords high specific sensitivity to the agent or substance evaluated in cytotoxicity tests, as they partly simulate the biological conditions under which such agents are used, and cell viability and functionality are the habitually evaluated parameters. While the use of established cell lines obtained from collections (EACC, ATCC, etc.) allows increased reproducibility of the results, they lack the specificity of primary cultures obtained from the affected tissues [Feigal et al.,1985; van

Pulp cell cultures for cytotoxicity tests

WyK et al., 2001]. In relation to this point, Louschall et al., 2002 likewise reported differences in cytotoxicity test results depending on the cell type used and on the test applied. In this sense, under identical conditions, the cells obtained from human tissues (pulp and mucosa) appeared to be more sensitive than established cell lines. However, in general, in the dental field, cell lines are more often used due to the ease with which they can be obtained; primary cell cultures and particularly pulp fibroblast or odontoblast cultures are less commonly employed [Wen Sun et al., 1990; Hill et al.,1991; Hashieh et al., 1999; Mitchell et al.,1999]. Hence the interest of developing a rapid and simple protocol for obtaining primary cultures of this cell type. It should be stressed that a single tooth yielded abundant biological material for testing in our study, with a low experimental variability, as all cells were originated from the same individual. Lastly, cell cultures clearly offer a rapid and extremely sensitive method for assessing the biocompatibility of a given material or substance. As regards pulp treatments in paediatric dentistry, the use of primary cultures would be adequate for assessing the biocompatibility of the agent used in application to pulpotomy, affording reliable results without the presence of uncontrolled variables such as marginal microleakage or contamination during the procedures carried out.

Conclusion Based on our results, we consider enzymatic digestion to be an effective and rapid technique for obtaining abundant biological material for cytotoxicity and biocompatibility studies of different dental materials.

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