Ameloblast
Each ameloblast is a columnar cell approximately 4 micrometers in diameter, 40 micrometers in length and is hexagonal in cross section. The secretory end of the ameloblast ends in a six-sided pyramid-like projection known as the Tomes' process. The angulation of the Tomes' process is significant in the orientation of enamel rods, the basic unit of tooth enamel. Distal terminal bars are junctional complexes that separate the Tomes' processes from ameloblast proper.
ameloblast
Ameloblasts are derived from oral epithelium tissue of ectodermal origin. Their differentiation from preameloblasts (whose origin is from inner enamel epithelium) is a result of signaling from the ectomesenchymal cells of the dental papilla. Initially the preameloblasts will differentiate into presecretory ameloblasts and then into secretory ameloblasts which lay down the tooth enamel. The differentiation from preameloblasts to ameloblasts occurs during the first stage of amelogenesis, called the pre-secretory (or inductive) phase. [1]
There are various factors which can affect the differentiation and development of ameloblasts, causing abnormalities to form within the tooth structure. One example is the BMP (bone morphogenetic protein,) which has an important role in ameloblast differentiation. When Follistatin, a BMP inhibitor, is over expressed in the epithelium of developing teeth, the ameloblasts do not differentiate and no enamel forms. Another example includes the conditional deletion of Dicer-1 in the epithelium of developing teeth may cause impaired differentiation of ameloblasts which results in deficient enamel formation. [2]
In this morphogenic stage the morphology of the cell is short, columnar with large oval nuclei. The golgi apparatus and centrioles are located in the proximal end of the ameloblast and mitochondria are dispersed throughout the cytoplasm.
In this stage the ameloblast cell become longer and the nucleus migrates towards the proximal end.In contrast to this, golgi apparatus and centrioles migrate towards the distal end.This change is referred to as "REVERSAL OF POLARITY" .During this stage odontoblast starts laying down dentin.
Reversal of Nutrition- as long as the ameloblast is in contact with the dental papilla it receives nutrient material from the blood vessels of the tissue but due to formation of this dentin the original source of nutrition is cut off and ameloblast is supplied by capillaries penetrating the outer enamel epithelium. This change in nutrition source is referred to as reversal of nutrition.
After the formation of enamel matrix mineralisation of enamel takes place which is known as maturation. During this stage the ameloblasts are slightly reduced in length. The stratum intermedium cells lose their cuboidal shape and assumed to be as spindle shape. During this stage ameloblasts also exhibits microvilli at their distal extremities.
Although adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study.
Stem cell-based tissue engineering has been proven a prospective approach to repair or replace an injured tissue or organ. Adult bone marrow stem cells (bone marrow stromal cells) are the first adult cell source capable of participating in tooth formation when confronted with the mouse embryonic dental epithelium that possesses odontogenic inducing capability [15]. At least five types of mesenchymal stem cells from adult human teeth have been isolated [16]. Among them, dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), and stem cells from the apical papilla (SCAP) could generate dentin/pulp-like complexes in ex-vivo culture [17,18,19]. Although these adult dental stem cells do not possess either odontogenic inducing capability or competence to support tooth formation when confronted with embryonic dental epithelia [20], they remain promising stem cell sources for regeneration of tooth mesenchymal components. On the other hand, the postnatal dental epithelium-derived stem cells are more difficult to obtain due to ameloblastic apoptosis during tooth eruption. It was reported that subcultured epithelial cell rests of Malassez can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage [21]. We and others have reported previously that nondental epithelia-derived human stem cells including human keratinocyte stem cells (hKSCs) [20, 22], gingival epithelial cells [23], and iPSCs [24], when recombined with either human or mouse embryonic dental mesenchyme, could support tooth formation and differentiate into enamel-secreting ameloblasts. However, less than 30% and 10% of these recombinant explants in subrenal culture formed teeth and produced enamel, respectively [22]. Such low efficiency of ameloblastic differentiation prevents use of these human stem cells as realistic cell sources for tooth replacement therapy. In addition, whether hKSC-derived dental epithelia exhibit an unusual life cycle and whether the regenerated enamel acquires the unique physicochemical characteristics remain elusive and warrant further exploration.
Studies indicated that either FGF8 or SHH alone is sufficient to promote limb regeneration in amphibian [25]. FGF8 or SHH is able to stimulate neurite outgrowth and cavernous nerve regeneration in vitro, respectively [26, 27]. In the tooth, FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program [28]. In this study, we developed an approach that greatly enhanced the ratio of ameloblastic differentiation of hKSCs and formation of tooth-like structures in tissue recombinants. We further examined the developmental process of differentiation of the hKSC-derived dental epithelium and present evidence for rapid differentiation of human ameloblasts and production of regenerated enamel with intact prisms the same as normal enamel. Meanwhile, we observed an increasing tendency for mineralization effect with improved mechanical properties in the regenerated enamel as cultivation extends. Our results provide a significant advance toward future use of human adult stem cells to generate implantable tooth organ ex vivo by tissue-engineering approaches.
Molar tooth germs dissected from surgically terminated human fetuses of 12th-week, 16th-week, and 19th-week gestation were provided by Fujian Province Maternal and Child Health Hospital. Use of human embryonic tissues in this study was approved by the Ethics Committee of Fujian Normal University, Fuzhou, China, and use of animals was approved by the Animal Use Committee of Fujian Normal University. Human fetus tooth germs and harvested recombinant samples treated with FGF8 and SHH protein prior to tissue recombination were fixed in 4% paraformaldehyde (PFA) overnight at 4 C on a rotator. Calcified tissues were further decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 1 week prior to being processed for dehydration and paraffin embedding. Sections were made at 10 μm, and were subjected to hematoxylin/eosin staining or Azan dichromic staining for histological analysis, and to immunohistochemical staining by antigen recovery technique. The following antibodies were used: anti-human ameloblastin, anti-human K18, anti-human p63, anti-human K10, anti-human integrin-β1 (Santa Cruz Biotech, Inc.), anti-human amelogenin, anti-human Sp3 (Abcam), anti-human Sp6, and anti-human Msx2 (HPA). For negative controls, the primary antibodies were omitted. Immunostaining, immunofluorescence, and TUNEL assay (Roche) procedures followed the instructions of the manufacturers. For western blot analysis, cultured hKSCs were extracted with urea lysis buffer. Equal amounts of samples were electrophoresed on 12% SDS polyacrylamide gels and transferred to NC membrane (Millipore). Immunoreactions were performed with the specific primary antibodies as mentioned earlier, visualized with fluorescent secondary antibodies (LI-COR), and scanned on an Odyssey Clx Imager (LI-COR). Blot images were quantified by densitometric analysis with ImageJ software.
In our previous report, we identified the human origin of hKSC-derived dental epithelial component and the mouse origin of the dental pulp with specific antibodies against human or mouse MHC antigen, respectively, in chimeric teeth to show no contamination of the mouse dental epithelial tissue in the recombinant experiment [22]. In the present study, we further recombined hKSCs with mouse dental mesenchyme genetically labeled with eGFP. Immunofluorescence studies indicated that no GFP-positive cells could be found in hKSC-derived ameloblasts that were marked with SP6 in chimeric teeth (Fig. 4J). In addition, we grafted E13.5 dental mesenchyme with removal of dental epithelium after enzyme treatment into nude mice for subrenal culture for 4 weeks as a further control. All 30 grafted samples either degenerated or formed tiny pieces of bone-like tissues (data not shown). These data provide more evidence to rule out the possibility of mouse dental epithelium contamination in the recombinant experiment.
Reciprocal heterotypic recombination of tissues of ectopic origin has been long used as a routine approach to study regulative interactions between tissue components in classical experimental embryology. Mammalian tooth development is dependent upon inductive interactions between epithelium and adjacent mesenchyme [45]. Both epithelial and mesenchymal components in tooth germ are indispensable for tooth development [46, 47]. Sequential and reciprocal interactions between the stomadial epithelium and the cranial neural crest-derived mesenchymal cells regulate tooth morphogenesis and differentiation. Odontogenic potential represents an instructive induction capability of a tissue to induce gene expression in an adjacent tissue and to initiate tooth formation, whereas odontogenic competence indicates the capability of a tissue to respond to odontogenic inducing signals and to support tooth formation. Tissue recombination experiments between isolated mouse molar epithelial and mesenchymal tissues have demonstrated that, during early tooth development, odontogenic potential resides first in the dental epithelium and then shifts to the mesenchyme [48, 49]. At the prebud stages of development (before and at E11.5), the presumptive dental epithelium possesses the potential to induce tooth formation in nondental mesenchyme. In contrast, at the early bud stage (E12.5) the odontogenic potential has switched to the mesenchyme, and this odontogenic mesenchyme is able to instruct nondental epithelium to form tooth-specific structures [48,49,50]. Our previous report demonstrated that such potential is also conserved in human embryonic dental mesenchymal tissues that are able to induce nondental epithelial tissues, such as human keratinocyte, and able to participate in tooth formation [20]. In-vitro bioengineering of primordial tooth germs represents a promising approach for tooth replacement therapy in the future [51]. Either in-vitro or ex-vivo generation of an implantable biotooth germ should follow the principles of tooth development. Based on this concept, previous studies including ours have demonstrated that human epithelium-derived stem cells, including iPSC-derived epithelium-like tissue, could be induced to participate in tooth formation when confronted with mouse dental mesenchyme with odontogenic potential. However, the efficiency of the induced ameloblastic differentiation of these cells was relatively low, and can be an obstacle for using adult stem cells as an epithelial cell source to develop tooth replacement therapy. In this study, in comparison with our previous report in which around 30% of tooth formation and 10% of ameloblastic differentiation were obtained [22], we achieved 70% and 100% of tooth formation and ameloblastic differentiation, respectively, by application of two key growth factors, FGF8 and SHH, in cell culture and tissue recombination, demonstrating that in the presence of appropriate odontogenic signals an efficient induction of enamel-secreting ameloblasts from hKSCs could be achieved. 041b061a72