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The current study aimed to prove that human dental pulp stem cells (hDPSCs) isolated from the pulp of third molars can show multilineage differentiation after cryopreservation. First, hDPSC were isolated via enzymatic procedures, and frozen in liquid nitrogen until use. After defrosting, cells were analyzed for proliferative potential and the expression of the stem cell marker STRO-1. Subsequently, cells were cultured in neurogenic, osteogenic/odontogenic, adipogenic, myogenic, and chondrogenic inductive media, and analyzed on basis of morphology, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR) for specific marker genes. All data were replicated, and the results of the primary cells were compared to similar tests with an additional primary dental pulp stem cell strain, obtained from the National Institutes of Health (NIH). Results showed that our cell population could be maintained for at least 25 passages. The existence of stem/ progenitor cells in both cell strains was proven by the STRO-1 staining. Under the influence of the 5 different media, both cell strains were capable to advance into all 5 differentiation pathways. Still differences between both strains were found. In general, our primary culture performed better in myogenic differentiation, while the externally obtained cells were superior in the odontogenic/osteogenic and chondrogenic differentiation pathways. In conclusion, the pulp tissue of the third molar may serve as a suitable source of multipotent stem cells for future tissue engineering strategies and cell-based therapies, even after cryopreservation.

The current study aimed to prove that human dental pulp stem cells (hDPSCs) isolated from the pulp of third molars can show multilineage differentiation after cryopreservation. First, hDPSC were isolated via enzymatic procedures, and frozen in liquid nitrogen until use. After defrosting, cells were analyzed for proliferative potential and the expression of the stem cell marker STRO-1. Subsequently, cells were cultured in neurogenic, osteogenic/odontogenic, adipogenic, myogenic, and chondrogenic inductive media, and analyzed on basis of morphology, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR) for specific marker genes. All data were replicated, and the results of the primary cells were compared to similar tests with an additional primary dental pulp stem cell strain, obtained from the National Institutes of Health (NIH). Results showed that our cell population could be maintained for at least 25 passages. The existence of stem/ progenitor cells in both cell strains was proven by the STRO-1 staining. Under the influence of the 5 different media, both cell strains were capable to advance into all 5 differentiation pathways. Still differences between both strains were found. In general, our primary culture performed better in myogenic differentiation, while the externally obtained cells were superior in the odontogenic/osteogenic and chondrogenic differentiation pathways. In conclusion, the pulp tissue of the third molar may serve as a suitable source of multipotent stem cells for future tissue engineering strategies and cell-based therapies, even after cryopreservation.

Pulp vitality is extremely important for the tooth viability, since it provides nutrition and acts as biosensor to detect pathogenic stimuli. In the dental clinic, most dental pulp infections are irreversible due to its anatomical position and organization. It is difficult for the body to eliminate the infection, which subsequently persists and worsens. The widely used strategy currently in the clinic is to partly or fully remove the contaminated pulp tissue, and fill and seal the void space with synthetic material. Over time, the pulpless tooth, now lacking proper blood supply and nervous system, becomes more vulnerable to injury. Recently, potential for successful pulp regeneration and revascularization therapies is increasing due to accumulated knowledge of stem cells, especially dental pulp stem cells. This paper will review current progress and feasible strategies for dental pulp regeneration and revascularization.

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The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic scaffold material, and then either cultured in an osteogenic medium or subcutaneously implanted into nude mice. For cell culture, samples were collected at weeks 0, 1, 3, and 5. Results were analyzed by measuring cell proliferation rate and alkaline phosphatase activity, scanning electron microscopy, and real-time PCR. Samples from the implantation study were retrieved after 5 and 10 weeks and evaluated by histology and real-time PCR. The results indicated that in vitro abundant cell growth and mineralization of extracellular matrix was observed for all types of cells. However, in vivo matured bone formation was found only in the samples seeded with rat bone marrow stromal cells. Real-time PCR suggested that the expression of Runx2 and the expression osteocalcin were important for the differentiation of bone marrow stromal cells, while dentin sialophosphoprotein contributed to the odontogenic differentiation. In conclusion, the limited hard tissue regeneration ability of dental pulp stromal cells questions their practical application for complete tooth regeneration. Repeated cell passaging may explain the reduction of the osteogenic ability of both bone- and dentinal-derived stem cells. Therefore, it is essential to develop new cell culture methods to harvest the desired cell numbers while not obliterating the osteogenic potential.

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An increasing number of investigations supports that adult stem cells have the potential to differentiate into matured cell types beyond their origin, a property defined as plasticity. Previously, the plasticity of stem cells derived from dental pulp (DPSC) has been confirmed by culturing cells in lineage-specific media in vitro. In the current study, the in vivo differentiation or maturation potential of DPSC was further analysed, by transplanting human DPSC/collagen scaffold constructs into subcutaneous tissue of immunocompromised mice. Cells received odontogenic, adipogenic or myogenic pre-induction, whereas control samples received no stimulation. Also blank collagen scaffolds were implanted. The results indicated that seeded cells produced tissue within the implanted constructs after 3 weeks of implantation. According to morphological and phenotypical changes, the pre-induced DPSC showed the ability to further differentiate along odontogenic, myogenic and adipogenic pathways in vivo. Moreover, DPSC without pre-treatment were able to spontaneously differentiate along odontogenic and adipogenic directions in vivo. However, only limited mature morphological changes were detected in histology. In summary, stem cells derived from human dental pulp form a suitable source for tissue engineering and cell-mediated therapy, although additional analyses should be considered.