| Teeth are composed of three hard tissues, and one soft tissue. The three hard tissues are the enamel, the dentin, and the cementum. The enamel is highly mineralized and is the hardest substance in the body. The enamel covers the crown of the tooth. The dentin, which makes up the bulk of the tooth, is softer than enamel but harder than bone. The tissue that covers the anatomic root of the tooth is cementum. The hardness of cementum is similar to that of the bone. The pulp is the only soft tissue of the tooth. It is almost fully encapsulated by dentin, and only connected with the environment through the narrow apical foramen, and by the openings of lateral canals. Even though, many pathological agents, such as caries, trauma, and periodontal disease, may cause the damage of dental tissues, or even cause teeth loss. Nowadays, numerous materials are developed to repair destructed dental tissues. However, most of these materials can only be used to restore the lost anatomical structure, but are insufficient to regenerate the biological functionality. Compare to endodontic treatment, tooth transplantation, or dental implant, a better result might be obtained by using tissue engineering techniques for dental tissue regeneration.Tissue can be regenerated in vivo or in vitro. The basic idea of tissue engineering is to seed isolated cells into a scaffold material. When the proper signaling molecules are used, cells can be driven towards the proper differentiation pathways, and the generation of specific tissue. In restorative procedures, such regenerated tissues can be used to replace the structure as well as functionality of the lost tissue. In general, the three key ingredients for tissue engineering are cells, signaling molecules, and scaffolds.Among the cell types that potentially can be used for tissue engineering, stem cells attract most of the attentions. A stem cell is relatively undifferentiated cell that has retained the ability to divide and proliferate throughout postnatal life. Stem cell populations provide progenitors, which in turn can differentiate into specialized cells. This property has made stem cell research become one of the most prominent scientific fields today. According to their origin, stem cells can be divided into embryonic stem cells (ES cells) and adult stem cells (AS cells). ES cells are the stem cells, which derive from the inner cell mass of blastocyst. ES cells are pluripotent, which means they can give rise to almost all cell types. Nevertheless, to obtain ES cells an embryo has to be dissected, which otherwise would have the potential to develop into a living being. Thus, research on human ES cells arouses enormous ethical debate. Consequently, most countries in the world carefully restrict research on ES cells, and some governmentseven fully prohibited the ES-related researches.Seen that situation, more and more researchers shifted their attentions towards adult stem cells. Adult stem cells are undifferentiated cell, which can be found in differentiated tissues or organs. AS cells, like ES cells, can renew themselves and also differentiate to yield specialized cells. At first, it was assumed that the differentiation ability of AS cells was restricted to the tissue in which the AS cells resided. However, resent researches have proven that AS cells also have the potential to form specialized cell types of the tissues they are not related with. This ability of AS cells is called transdifferentiation or plasticity. This founding drastically increases possible application of AS cells. With increasing researches of stem cell, the presence AS cells has been confirmed in many differentiated tissues, including in some tissues which once seemed unlikely, like brain tissue.The dental pulp contains various types of cells. Among those, the most characteristic cell type is odontoblast. Odontoblasts can synthesize dentin during their entire lifespan. However, odontoblasts are very sensitive. There are easily destroyed, and loss the dentin-producing function when over stimulated. Odontoblasts are post-mitotic cells, and cannot enter the cell division circle again. Accordingly, the formation of reparative dentin suggests the existence of special cell type within dental pulp, which can differentiate into odontoblasts and compensate their function.A research group from NIH has isolated adult stem cells from postnatal human dental pulp. It has been proven that those cells also share the two basic characteristics of stem cell: i.e. self-renewal, and differentiate potential. This cell line NIH developed has been named postnatal human dental pulp stem cells (hDPSCs). After being seeded into immunocompromised mice, hDPSCs were able to synthesis dentin pulp complex-like tissue. Although the mechanism of the differentiation is still vague; this research suggests the possibility of repairing dental tissue by using autologous dental pulp stem cells.It also has been proven that hDPSCs have the potential to differentiate into nerve cells and adipocytes. These results suggest that the human dental pulp stem cells show multilineage differentiation ability. However, no systematic research on this particular ability of human dental pulp stem cells has been reported until now.One of the most commonly used human AS cells are bone marrow mesenchymal stem cells (BMMS cells). BMMS cells have the capability to produce themselves and to differentiate into several specialized cell types. BMMS cells usually are isolated by a bone marrow aspiration from the posterior iliac crest. However, bone marrow aspiration is an uncomfortable procedure. Furthermore, the person who receives the operationpossibility can get sequelae after operation, such as a post-operative infection.For human beings, the third molars are easily get impacted, because in evolution the jawbones of human beings were getting smaller. Most impacted third molars need to be extracted. Because of their low mastication efficiency, removal of a third molar will not influence health, even when it is erupted in the right position. If we could prove that human dental pulp stem cells can be obtained from third molars, and show plasticity even after cytopreservation, the dental pulp from third molars might serve as a novel source of stem cells that can be used for autologous tissue regeneration.Next to the cell source, also the choice of the scaffold material is significant for tissue engineering. Materials should be designed according to the properties of the tissue replaced. The basic criteria of an ideal material include: 1) Stable physical and chemical properties; 2) Not cytotoxic; 3) No induction of immunoreaction or inflammation; 4) Degradable; 5) Appropriate mechanical strength; 6) Sufficient manipulability. Of course, no single material can meet with all these requirements. Two of the most commonly used materials for bone regeneration are titanium, and ceramics. Titanium is non-degradable; and the degradation properties of ceramic are quite limited. Still, they are widely used for hard tissue regeneration, because both materials are highly biocompatible, and support the hard tissue regeneration. For the current research also these two materials were chosen, as the hard tissues of teeth share similar physical properties with the bone tissue.In this research, first the existence of stem cells, or progenitors, in the isolated rat dental pulp has been proven. Then, the ability of such cells was analyzed, including their abilities to differentiate into odontoblast-like cells and to form calcified extracellular matrix. Second, human dental pulp stem cells were isolated; and the capability of multilineage differentiation has been evaluated. Consequently, this research contains two main parts:Part One: Differentiation ability of rat dental pulp cells in vitro.First, we isolated a group of dental pulp cells from 40-day-old Wistar rat, by enzymatic digestion. STRO-1 is a surface marker for progenitor cells from mesenchym; and has been used to separate bone marrow stem cells. Similarly, the expression of STRO-1 on rat dental pulp cells was confirmed by immunohistochemical staining. Approximately 5% of the rat dental pulp cells were STRO-1 positive, which suggested the existence of stem cells or progenitors in the isolated rat dental pulp cells.After cytopreservation, rat dental pulp cells were seeded on 24-well plates in osteogenic culture medium, to analyze their differentiation ability on two-dimension(2-D) smooth culture surface. Cell proliferation, alkaline prosphatase (ALP) activity, and calcium contend were measured to assess cell growth behavior. The results showed the growth behavior of rat dental pulp cells was similar to that of rat bone marrow stem cells cultured under similar conditions. Von Kossa staining confirmed the rat dental pulp cells were able to produce mineralized nodules. Scanning electron microscopy (SEM) was used to get a better view of cell morphology and the formed nodules. Energy dispersive spectroseopy (EDS) analysis demonstrated the chemical components of the nodules were calcium and phosphate, with a Ca:P ratio of 1.4. RT-PCR verified the expression of dentin sialophosphoprotein (DSPP), a specific protein of odontoblasts, after culture for 5 days in osteogenic medium. All those results together confirmed the isolated rat dental pulp cells had retained their differentiation ability after cytopreservation.In addition, also the differentiation ability of rat dental pulp cells was analyzed when the cells were cultured on 3-D scaffolds. Two materials were selected in this experiment, a porous hydroxyapatite tricalcium phosphate (HA/TCP) ceramic and fibrous titanium mesh scaffold. Cells were seeded in the two kinds of scaffolds and the formed cell-scaffold constructs were cultured in osteogenic medium. Analyzed parameters were the same as those used for 2D cultures. Their results of the experiments on 3D scaffold showed similar results as the 2D cultures. However, in comparison the cells were able to maintain longer on 3D scaffolds; the peak of ALP activity was postponded; and Ca:P ratio was higher. The Ca:P ratio of nodules formed on titanium fibre meshes after 8 weeks of culture was 1.6-1.8. This ratio is equivalent to that of natural dentin and hydroxyapatite materials. The behaviors of rat dental pulp cells was analogous on both materials, meaning that the odontogenic properties of rat dental pulp cells were supported equally well by both materials.These results further confirmed that there were stem cells or progenitors in the isolated rat dental pulp cells, and they were able to differentiate into odontoblast-like cells.Part 2 Isolation of human dental pulp stem cells, and assessment of the differentiation ability in vitro.At first, a group of postnatal human dental pulp cells, with a high proliferation rate was isolated. Some of the cells showed positive staining of antibody against STRO-1. The cells maintained proliferating in a stable manner for more than 20 population doublings. These results implied the existence of stem cells or progenitors in the isolated cell population. The group of cell was named human dental pulp stromalcells-NB (hDPSC-NB).Then, the multilineage differentiation abilities of such cells were evaluated, including neurogenic, odontogenic, adipogenic, myogenic, and chondrogenic pathways. The basic method applied was to culture the cells in different inductive media, and subsequently analyze cells on the basis of morphology, immunohistochemical staining, and RT-PCR.Two types of cells were used in this research. Next to the hDPSC-NB, hDPSC-NIH cells were kindly provided by Dr. Shi from the NIH. hDPSC-NIH is a cell line isolated in the NIH research group. As mentioned above, it is already proven that the hDPSC-NIH line contains human dental pulp stem cells.After culture in neurogenic inductive medium, both types of postnatal hDPSCs showed a typical neuronal morphology. Also, both expressed Neuronal nuclei protein (NeuN) and Neuron specific enolase (NSE). Those results suggested human dental pulp stem cells had the potential to different into neuron-like cells.Similarly, after incubation in osteogenic/odontogenic medium, both postnatal hDPSCs showed the expression of DSPP, the specific protein of odontoblasts. However, no typical column-shaped cells were observed. Nonetheless, the results supported the idea that human dental pulp stem cells were able to differentiate into odontoblast-like cells.After adipogenic differentiation, part of the cells increased in size. However, no lipid-containing droplets were found. Still, the expression of adipocyte-specific protein, peroxisome-proliferating activated receptor y 2 (PPARy2) and glucose transporter-4 (GLUT4), implied that the human dental pulp stem cells differentiated into adipocyte-like cells. Again, both of the cells types used showed similar results.Under the influence of myogenic media, some cells elongated and showed a myofibre-like morphology. However, no cells with multiple nuclei were observed. The present of myoblast determination protein 1 (MyoDl), a specific protein of myoblast, was confirmed both by immunohistochemistry and RT-PCR. Immuno-analysis also showed the expression of myosin heavy chain (MHC). Both of the human dental pulp stem cells gave analogous results.Finally, both types of human dental pulp stem cells were cultured in chondrogenic medium. During this part of the experiment, a "pellet culture" method was used. After 4 weeks, the formed structures exhibited a cartilage-like appearance. The excellular matrix (ECM) showed positive staining for alcian blue, which proved the existence of sulfated proteoglycans within the ECM. The present of Collagen type II in ECM was verified by immunoanalysis. The production of Collagen type II and sulfatedproteoglycans are specific parameters for chondrogenic differentiation. In comparison, the tissue formed by hDPSC-NIH contained more differentiated cell, and showed more distinct cartilage lacuna-like structures. In other words, the latter morphology resembled more the appearance of bone marrow stem cells after chondrogenic differentiation.In conclusion, human dental pulp stem cells have the ability of multilineage differentiation. The pulp of third molar may be a novel source of multipotent stem cells for future tissue engineering strategies, and cell-based therapies.
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