Methods: Fifteen days after birth rat pups received sub-dermal injections superficial to the posterior frontal suture. Rats were randomly assigned to one of three groups and injected with: 1) PBS (Sham Control), 2) collagen gel vehicle with Tgf-beta3 plasmid, 3) collagen gel vehicle with carrier plasmid, pCMV6-xl5. Ten days after injection, rats were euthanized by CO2 narcosis and the posterior frontal sutures were harvested for histological examination. Briefly, tissues were fixed, decalcified and paraffin-embedded, then sectioned at 6 µm using the coronal suture as a landmark. The extent of bony bridging at the sutures was measured at progressive distances anterior to the coronal suture. Percent bridging was calculated as bridging height divided by suture height x 100. Percent bridging were measured from sections every 6 µm beginning at 600 µm from the coronal suture and extending 900 µm anteriorly. Statistical analysis was performed using a one-way analysis of variance (ANOVA) comparing percent suture fusion in each group. Post hoc tests were performed using the Tukey-Kramer multiple comparison test with p< 0.05 considered significant.
Results: In all animals bridging began on the endocranial side and extended ectocranially. In animals implanted with collagen gel containing Tgf-beta 3 plasmid, a statistically significant reduction in mean percent bridging of the posterior frontal suture was seen (p<0.05) as compared to animals implanted with either PBS or the carrier plasmid.
Conclusions: The above results indicate that an increase in plasmid-encoded Tgf-beta3 was effective in maintaining the patency of rat posterior frontal sutures in vivo. This study supports the important role of Tgf-beta3 in cranial suture fusion and its possible clinical applications in the prevention of re-ossification following surgical treatment of craniosynostosis.