Localized Infusion of Tunicamycin in Rat Hemimandibles: Alteration of the Basal Lamina Associated with Maturation Stage Ameloblasts

Abstract

S U M M A R YAt the beginning of the maturation stage of amelogenesis, ameloblasts deposit a basal lamina (BL) at the interface between their apical surface and maturing enamel. This structure is rich in glycoconjugates and is proposed to exhibit adhesive and/or filtering functions. To clarify its role, we have applied a recently developed surgical window model to locally administer tunicamycin (TM), an antibiotic that interferes with N -glycosylation, in the rat hemimandible using an osmotic minipump. Male Wistar rats were infused with either TM or saline as a control. Lectin-gold cytochemistry was performed to reveal glycoconjugates in the BL. Immunogold labeling of enamel proteins and albumin was carried out to verify whether depletion of N -linked sugars in the BL affects the content and distribution of endogenous and exogenous proteins in the enamel layer. Under the influence of the drug, the BL became irregular and exhibited alterations in structural organization and composition. The number of Helix pomatia agglutinin binding sites was not significantly affected but their distribution was altered. The labeling density of wheat germ agglutinin over the BL was slightly reduced. Immunoreactivity for enamel proteins showed only a small decrease, but that of albumin, both between ameloblasts and within the enamel layer, increased significantly. No structural alterations were observed in the contralateral incisor and in other sampled tissues and organs. These results demonstrate that it is possible to achieve a localized administration of TM without systemic side effects and lend support to the proposal that the BL represents a specialized structure with filtering functions.

Odontogenic ameloblast-associated and amelotin are novel basal lamina components

“…In addition, local administration of tunicamycin in rat incisors, an antibiotic that interferes with N-glycosylation of proteins, was shown to cause alterations of its structure. These alterations were not enough to cause detachment of the enamel organ, but there was an abnormal presence of albumin in maturing enamel, suggesting that this atypical BL may exhibit filtering functions (Orsini et al 2001).…”

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section

“…In order to probe further function of matrix proteins in bones and teeth, we have developed a bony window system to carry out various experimental manipulations, in the context of the whole animal biology (Vu et al 1999;Orsini et al 2001;Nanci et al 2004;Wazen et al 2007). The combination of colloidal gold immunolabeling and the bony window has allowed us to uniquely look at whether circulating NCPs can integrate the same sites where vectorially secreted NCPs normally incorporate, thereby participating in a similar manner to mineralization events.…”

“…Such large dosages rarely are physiological. Our laboratory has developed an experimental system that allows the controlled administration of biological and chemical agents through a surgically-created window in rodent bones (Vu et al 1999;Orsini et al 2001;Wazen et al 2006). Administration of DNP-tagged molecules through surgically created holes in bones permits tracer experiments to be carried out at near physiological concentrations, to distinguish the administered molecules from endogenous ones, and to follow them over time.…”

“…We have infused tunicamycin through a bony window in the rat hemimandible to study the role of glycoproteins on the assembly and function of this basal lamina (Orsini et al 2001). Tunicamycin is an antibiotic that interferes with Nglycosylation and that has significant side effects when used systemically.…”

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector

“…The laboratory has been using 'bony windows' coupled to osmotic minipumps to continuously deliver to calcified tissues bioactive agents. [37][38][39] Whereas this approach allows administration of small quantities that respect the local tissue physiology, it also permits to locally attain relatively high concentrations and, most importantly, to continuously replenish any material that would be rapidly cleared away.…”