Original ArticleEstablishment of ex vivo mucocele model using salivary gland organ culture
Introduction
Minor salivary glands are found in most parts of the oral cavity except the gingiva. Mucoceles, which are generally asymptomatic, are a common oral mucosal lesion that originate from those glands, and are seen more frequently in children and adolescents [1], [2]. The term mucocele describes the accumulation of mucus within a salivary gland, and they are typically subdivided into two types based on histopathological findings. One is a mucous extravasation cyst, which is generally regarded as being the result of trauma, such as lip biting, while the other is a mucus retention cyst caused by the obstruction of the duct of a minor or accessory salivary gland [3], [4]. In the retention type (also called salivary duct cyst), trapped mucus is lined by columnar or cuboidal ductal epithelium [3], [5].
The clinical features of mucoceles have been well documented and vary depending on depth within soft tissue and the degree of keratinization of the overlying mucosa. Superficial lesions are presented as raised soft-tissue swelling with a bluish color, while deeper lesions are more nodular, lack a vesicular appearance, and have a normal mucosal color. Size can vary from 1 mm to several centimeters [4]. The lower labial mucosa is the most frequently affected site, though mucoceles can also develop in the cheek, tongue, palate, and floor of the mouth, where it is called a ranula [3], [6]. Mucoceles often arise within a few days after minor trauma, but then plateau in size and can persist unchanged for months unless treated [7], [8]. Although most cases are asymptomatic, functional disturbances such as difficulty with eating and changed dietary habits may require immediate surgical intervention. Different techniques have been described for the treatment of mucoceles, though they usually require surgical excision [8], [9]. On the other hand, no evidence showing the mechanism of formation or healing of a mucocele has been reported.
Recently, salivary gland organ cultures have been used to analyze the molecular mechanism of branching morphogenesis. In vivo, mouse submandibular gland (SMG) development is initiated as a thickening of oral epithelium on about embryonic day 12 (E12), when an initial epithelial bud on a stalk grows into condensing neural crest-derived mesenchyme. Clefts in the epithelium result in 3–5 epithelial buds by E13.5, and then branching morphogenesis occurs with continued proliferation, along with successive rounds of cleft formation, duct elongation, and lumen formation, resulting in a highly branched gland by E14. [10] Ex vivo organ cultures of SMG tissues from E13 show a similar developmental pattern of branching morphogenesis on the filter as compared with in vivo development. Previously, our group identified the role of platelet-derived growth factors (PDGFs) and their receptors in SMG branching morphogenesis [11]. PDGFs induced fibroblast growth factor 7 (FGF7) and FGF10 expression in SMG mesenchyme, which in turn enhanced the branching of organ-cultured SMG tissues [11], [12], indicating that epithelial–mesenchymal interaction via PDGF–FGF signaling is important for salivary gland development and further that this culture system is useful to identify the mechanism of salivary gland morphogenesis. Based on the results of those experiments, we considered that the pathogenic condition of salivary glands could be reproducible using this organ culture system. For the present study, we established a mucocele pathogenic model using SMG organ cultures.
Section snippets
Animals and reagents
Pregnant ICR mice were used in this study. Dulbecco's modified Eagle's medium/F-12 (D-MEM/F-12, Gibco), penicillin and streptomycin (Pen Strep, Gibco), vitamin C (Sigma–Aldrich), and a transferrin agent (Sigma–Aldrich) were used for the organ cultures.
Ex vivo submandibular organ cultures
Submandibular and sublingual salivary gland rudiments (referred to as SMGs in this study) were dissected from E13 mice and cultured on cell culture inserts (0.4 μm pore size, Falcon) at the air–medium interface. Each insert was placed in 2 ml of
Establishment of mucocele by removal of upper medium
Submandibular and sublingual salivary gland organs from E13 mouse embryos (Fig. 1A) were digested and cultured on filter inserts, with 20 μl of culture medium added during the culture (Fig. 1B). After 48 h, the upper medium was removed with a pipette as much as possible (Fig. 1C). At 7 days, mucus began to be restored in the salivary gland ducts of the upper-medium-removed group. Furthermore, seven of the 22 (31.8%) specimens in the upper-medium-removed group showed mucocele-like mucus
Discussion
In the present study, we established an ex vivo model of mucocele development by removing the upper medium from salivary gland organ cultures. This is the first report of a salivary gland pathogenic condition being developed ex vivo, except for genetic disorders such as aplasia of the lacrimal and salivary glands and lacrimo-auriculo-dento-digital (LADD) syndrome, which is characterized by aplasia or hypoplasia of the salivary and lacrimal systems [13].
In our previous study, we analyzed the
Conclusion
This is the first study to establish mucus retention type mucoceles using ex vivo mouse salivary gland organ cultures. Mucoceles developed after the removal of the upper medium in about 60% of the ex vivo cultures. We consider this model useful to analyze the effects of mucus retention on salivary gland development and function, as well as for the establishment of treatments for mucoceles including drug usage and surgical procedures.
Conflict of interest
None of the authors have any conflicts of interest that should be disclosed.
Acknowledgments
This work was supported by grants-in-aid (20679006 to S. F.) from the Ministry of Education, Science, and Culture of Japan, and the NEXT Program (LS010 to S. F.).
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Cited by (1)
Identification and function analysis of ameloblast differentiation-related molecules using mouse incisors
2020, Pediatric Dental JournalCitation Excerpt :So, abnormal epithelium may induce mesenchymal disorder. These interactions between mesenchyme and epithelium that occur during tooth development are common to the development of all organs, including the salivary glands [6,7] and hair [8]. Tooth development studies may lead to elucidation of the differentiation process of various ectodermal tissues.
- 1
These two authors contributed equally to this work.