Oral mucositis is a frequent, often dose-limiting early adverse effect of head and neck cancer radio(chemo)therapy [
1,
2], especially when the oral cavity and major salivary glands are in the radiotherapy treatment field [
3]. The epithelium of the oral cavity is well suited for its many unique functions, having regional differences in the keratinization of the mucosa [
4]. Aside from differences in keratinization, there are regional differences in tissue thickness and cell turnover time. Epithelial homeostasis requires that the rate of cell production equals the rate of desquamation. This concept is known as turnover time [
4]. Turnover time also dictates the rate of healing. The balance between epithelial cell production and desquamation has important implications for mucosal health and disease [
5]. Proliferation is based on tissue-specific stem cells, which represent a fraction (of unknown size) of the proliferating cells. Stem cells physiologically divide into one new stem cell (a self-renewing system) and one non-stem daughter cell. The latter can, as a transit cell, undergo a limited number of cell divisions before terminal differentiation and loss at the surface [
6]. The cell production, either from stem cell proliferation or transit cell division, is limited to the basal layer and the deeper parts of the spinos layer; these are depicted as a germinal layer (stratum germinativum). The functional epithelial layer contains the residual part of spinos layer and the granular layer, which is characterized by basophilic keratohyalin containing granules [
7]. During differentiation, cells increase in size and flatten towards the superficial layer. A finely regulated differentiation program process is characterized by the sequential expression of different proteins, coincident with the phenotypic evolution from basal cell to the mature, nonviable stratum layer [
8]. The superficial layer is composed of the keratin layer where the keratohyalin is converted in keratin and displays final differentiation with complete keratinization [
9,
10]. Murine oral mucosa presents as a multilayered squamous epithelium composed of a germinal layer, a functional layer, and a superficial layer, and is largely comparable to human oral mucosa [
11]. Taken together, the oral mucosa represents a perfectly suited model for studying proliferation and differentiation. However, the molecular mechanisms governing mucosal differentiation are still largely unknown [
8]. Typical early IR effects are found in turnover tissues, where physiologically permanent cell loss from the differentiated, post-mitotic compartments of the tissue is well balanced by proliferation in the germinal parts of the tissue [
12,
13]. Following mechanical or chemical interactions, superficial cell loss triggers cell production in the germinal layer [
14]. As a consequence of the proliferative impairment, the reduced cellular supply to the differentiated tissue layers results in progressive hypoplasia and, eventually, incomplete cell depletion [
15]. The molecular pathogenesis of this ulcerative epithelial process is still unclear, but the involvement of changes in epithelial differentiation processes is highly likely. DS plays an important role in wound healing, like other glycosaminoglycans (GAG), and it binds to fibroblast growth factor (FGF)-2, which stimulates cell proliferation in response to injury [
16]. FGF‑2 has been reported to be connected to DS and activated by DS [
17], and it functions as a mitogen that signals mesenchymal cell migration, proliferation, and differentiation [
5]. Furthermore, the underlying mechanisms of DS’ mucositis-ameliorating action may include modulation of epithelial differentiation, which needs to be defined [
16]. The aim of the present study, therefore, is to investigate and characterize oral epithelial differentiation process during daily fractionated IR alone and in combination with DS treatment in the same well-established mouse model.