ABSTRACT
The in vitro three-dimensional sphere model has already been established as an important tool in fundamental sciences. This model facilitates the study of a variety of biological processes including stem cell/niche functions and tissue responses to injury and drugs. Here we describe the complete protocol for the in vitro formation of spheres originated from the epithelium of rodent incisors. In addition, we show that in these spheres cell proliferation is maintained, as well as the expression of several key molecules characterizing stem cells such as Sox2 and p63. These epithelial dentospheres could be used as an in vitro model system for stem cell research purposes.
ABSTRACT
Cell-based tissue regeneration is an attractive approach that complements traditional surgical techniques for replacement of injured and lost tissues. The continuously growing rodent incisor provides an excellent model system for investigating cellular and molecular mechanisms that underlie tooth renewal and regeneration. An active population of dental epithelial progenitor/stem cells located at the posterior part of the incisor, commonly called cervical loop area, ensures the continuous supply of cells that are responsible for the secretion of enamel matrix. To explore the potential of these epithelial cells in therapeutic approaches dealing with enamel defects, we have developed a new method for their in vivo administration in the posterior part of the incisor. Here, we provide the step-by-step protocol for the isolation of dental epithelial stem cells and their delivery at targeted areas of the jaw. This simple and yet powerful protocol, consisting in drilling a hole in the mandibular bone, in close proximity to the cervical loop area of the incisor, followed up by injection of stem cells, is feasible, reliable, and effective. This in vivo approach opens new horizons and possibilities for cellular therapies involving pathological and injured dental tissues.
