Isolation of Stem Cells from Pulp Deciduous Teeth Dog

Stem Cells (SC) have the potential for self-renewal and differentiation. And some research groups used cell therapy to regenerate lost or injured tissues. With that some research groups to use in cell therapy to regenerate lost or injured tissues. The isolation of SC can be performed in various tissue origins. This paper aims to describe a protocol for isolation of stem cells from the pulp of deciduous teeth dog.


Introduction
The Stem Cells (SC) are defined as cells capable of selfrenewal and tissue differentiation.They are present in all tissues [1,2] and can be obtained from endodermal, mesodermal and ectodermal tissues [3].Stem cells have been isolated from: bone marrow, neural tissue, skin, retina and human dental pulp [1,4].
The Stem Cells (SC) are classified according to origin in embryonic/fetal or adult/postnatal.The difference between them is in plasticity, or the potential to produce different specialized cell lines.Since embryonic have great plasticity, but their use is still surrounded by ethical and legal issues [1].
Early studies with human SC were made with pulp tissue of extracted third molars [5] teeth.The discovery of SC dental pulp and the advancement of cellular and molecular biology led to the development of new regenerative therapies [6].The pulp stem cells from permanent or deciduous teeth are able to provide cells for clinical application [5].In dentistry, tissue engineering explores the SC primary or permanent teeth [7].
The possibility of using stem cells to regenerate the periodontium has motivated researchers [8] because periodontal regeneration represents a major breakthrough in periodontal therapy [9].
As Lin et al. [8], cells in bone marrow and adipose tissue regenerate alveolar bone and form a structure similar to the periodontal ligament.Tissue engineering has been aided by animal studies showing positive results [10].for 5 minutes at room temperature.The pellet was re suspended in DEMEM/HEPES (Gibco) culture medium, supplemented with 10% fetal bovine serum (Gibco), 100 units/ml penicillin, 100 μg/ ml streptomycin (Gibco) and 3.7 mg/L HEPES (Sigma-Aldrich).Centrifuged again at 800 g for 5 minutes, while the medium was discarded and the resulting cell suspension was seeded into one well of a 6 well plate.The exchange of culture medium was after 24 hours of the initial plating and after every 2-3 days.The culture was maintained under these conditions until greater than 80% confluence (Figure 2), when it held its first passage confluence.In transplants, the cells in culture were harvested with a solution of 0.5% trypsin-EDTA (Sigma-Aldrich) and transferred to subcultures in the respective culture medium.The subculture was maintained in monolayer until its next peal was necessary.Cultures of stem cells were transplanted up to 8 times.

Results and Discussion
Isolation and culture of pulp tissue was considered positive, since 80% of the samples showed cell growth after 24 hours of cultivation.Luisi et al. [11] observed a similar result.
The methodology for storage and transport of biological material from the time it was collected until processing differs from Bernardi et al. [12], but proved to be efficient regarding contamination, since none of the cell cultures was affected.It is believed that the prior training of the team was determinant to the result.Access to dental pulp was safe and effective only with the use of alveolotomo while Bernardi [13] made use of high rotation and drills, which is not always present in lab equipment.Unlike Miura et al. [14] who added to this dispase solution.With this, protocol becomes more practical, less expensive and is also financially efficient for cell proliferation.
In considering the description of Peng et al. [8] about the easy and rapid expansion of stem cells from the dental pulp of human deciduous tooth in vitro, we can mention that the dental pulp of deciduous teeth of dogs had similar behavior, because it got the proper confluence mobile ringing in the shortest time quoted in the literature [11].
Meeting isolation and cell culture are requirements to work with regeneration of tissue when using cell therapy [8].The protocol suggested in this paper can help new groups that have an interest in this research line.

Conclusion
The protocol suggested by our group is efficient for the isolation of dental pulp of deciduous teeth of dogs and features a lower financial cost if the methodology used by other research groups to obtain stem cells were purchased.

Figure 1 :
Figure 1: Deciduous tooth from which the pulp was obtained for cell culture.