Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Abstract

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected. Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues. Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation. Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo. On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Ameloblastomas Exhibit Stem Cell Potential, Possess Neurotrophic Properties, and Establish Connections with Trigeminal Neurons

“…Microfluidic devices were prepared as previously described [36,37]. Glass coverslips were coated overnight at 37 • C with 0.1 mg/mL poly-D-lysine and stored in 70% Ethanol at 4 • C. Polydimethylsiloxane (PDMS) microfluidic devices (Millipore A150, Switzerland, 2 cm × 2 cm) were punched with a 1 mm-diameter biopsy punch on the neuronal side to enable the insertion of the trigeminal ganglion and then sterilized with 70% ethanol.…”

“…Trigeminal ganglia were placed through the punched hole into the neuronal chamber of the microfluidic device [36,37]. Ganglia were cultured alone for 5-7 days, until axons were projected into the stem cells chamber.…”

“…We then probed the neurotrophic properties of ameloblastoma cells in a microfluidic co-culture system ( Figure 6A) [6,36,37]. In this system we co-cultured whole trigeminal ganglia and AB10 cells ( Figure 6A).…”

Innovative Dental Stem Cell-Based Research Approaches: The Future of Dentistry

“…Several in vivo and in vitro studies have provided important information about the patterns of innervation of dental tissues in different animal models as well as the main molecules involved in nerve trophic support, repulsion, and attraction [ 10 , 53 – 56 ]. To date, little information is available concerning the effects of innervation on human dental stem cells [ 57 ]. Cocultures have been used and constitute a valuable method to investigate and manipulate the interactions between nerve fibers and target tissues of human origin in a controlled and isolated environment ( Figure 2 ) [ 57 , 58 ].…”

“…To date, little information is available concerning the effects of innervation on human dental stem cells [ 57 ]. Cocultures have been used and constitute a valuable method to investigate and manipulate the interactions between nerve fibers and target tissues of human origin in a controlled and isolated environment ( Figure 2 ) [ 57 , 58 ]. The use of these and other state-of-the-art techniques, in combination with in vivo approaches in animal models, will allow gaining fundamental knowledge regarding the role of innervation in stem cell behavior and the mechanisms that drive tooth reinnervation [ 6 ].…”