Requirement of alveolar bone formation for eruption of rat molars.

Tooth eruption is a localized event that requires a dental follicle (DF) to regulate the resorption of alveolar bone to form an eruption pathway. During the intra-osseous phase of eruption, the tooth moves through this pathway. The mechanism or motive force that propels the tooth through this pathway is controversial but many studies have shown that alveolar bone growth at the base of the crypt occurs during eruption. To determine if this bone growth (osteogenesis) was causal, experiments were designed in which the expression of an osteogenic gene in the DF, bone morphogenetic protein-6 (Bmp6), was inhibited by injection of the first mandibular molar of the rat with a small interfering RNA (siRNA) targeted against Bmp6. The injection was followed by electroporation to promote uptake of the siRNA. In 45 first molars injected, eruption was either delayed or completely inhibited (seven molars). In the impacted molars, an eruption pathway formed but bone growth at the base of the crypt was greatly reduced compared with the erupted first-molar controls. These studies show that alveolar bone growth at the base of the crypt is required for tooth eruption and that Bmp6 may be essential for promoting this growth.

Electroporation to deliver plasmid DNA into rat dental tissues.

BACKGROUND: Delivery of DNA into the target tissues is an important technique in gene function studies and gene therapy. Surgical treatment of tooth eruption disorders, such as impacted third molars, is a major healthcare cost. Because the dental follicle (DF) is essential for regulating tooth eruption, establishment of local gene transfer protocols is needed to determine the effect of various genes on eruption and to develop gene therapy approaches for inducing the eruption of impacted molars. METHODS: Plasmids containing lacZ reporter gene were injected into rat mandibles and then electroporated at the designated settings. Mandibles were collected 24 h after electroporation for X-gal staining to evaluate the transfection efficiency. Tissues were collected at various days post-electroporation to determine the expression of the transgene. RESULTS: For the DF, depth of injection and pulse number appear to be important. Six pulses can achieve above 80% transfection of the DF at 50 V or 120 V. For alveolar bone (AB) transfection, voltages are important, with 120 V being optimal. Regarding pulse durations, we determined that durations of 20 and 30 ms achieve the maximum transfection in AB and DF, respectively. CONCLUSIONS: The present study demonstrates for the first time the feasibility of electroporation to locally deliver plasmids into dental tissues. Parameters affecting electroporation to deliver plasmids into the dental tissues were optimized. This protocol could be used to deliver short hairpin RNA or genes of interest into the dental tissues to regulate tooth eruption. Thus, it may be possible to develop nonsurgical treatments for inducing the eruption of impacted teeth.

In vivo expression of RANKL in the rat dental follicle as determined by laser capture microdissection.

Tooth eruption is a localized event in which many of the genes required for eruption are expressed in the dental follicle. A major function of the follicle is to recruit mononuclear cells for osteoclastogenesis such that the alveolar bone can be resorbed. Osteoclastogenesis is primarily regulated by receptor activator of nuclear factor-kappa B ligand (RANKL), colony-stimulating factor-one (CSF-1) and osteoprotegerin (OPG). In the rat first mandibular molar, osteoclastogenesis is maximal at day 3 and CSF-1 is maximally expressed in the follicle at this time whereas OPG expression is reduced. Whether or not RANKL is expressed in vivo in the follicle is controversial, however. It is critical to determine this because others have shown that in partially-rescued mice null for RANKL, teeth do not erupt. This suggests that RANKL should be expressed in the follicle for eruption to occur. Thus, to precisely determine if RANKL is expressed in the follicle in vivo, laser capture microdissection (LCM) was used to excise dental follicle tissue from frozen sections followed by RNA isolation and RT-PCR. The results show that RANKL is expressed in the dental follicle at days 1-9 postnatally. The technique was confirmed by controls showing that LCM isolates of the follicle, and alveolar bone, express OPG. Also, LCM isolates of alveolar bone were positive for RANKL. Thus, RANKL has now been shown to be expressed in the follicle and it is probable that interactions between it, CSF-1 and OPG regulate locally the osteoclastogenesis needed for tooth eruption.