Immunohistochemical analyses of bioengineered tooth tissues

Objective: The objective of this research was to characterize the tooth tissues of 25-week and 30-week old tissue engineered tooth implants. Methods: Biodegradable scaffolds in the shape of human incisors were fabricated, seeded with dissociated porcine third molar cells and organoids, and implanted into the omentum of athymic Rowett rats where they were allowed to develop over 20 to 30 weeks. Immunohistochemistry of paraffin embedded bioengineered tooth tissue sections was performed by use of affinity-purified polyclonal anti-amelogenin or anti-collagen type I antibodies in conjunction with biotinylated secondary antibodies and a streptavidin-horse radish peroxidase detection system. Results: Within the engineered tooth tissues amelogenin was detected along the secretory edge of ameloblasts, within ameloblast cells, and within the enamel with the most prominent staining near the dentin-enamel junction. Collagen type I was detected within the dentin tubules and odontoblast cells of the bioengineered tooth tissues. Conclusions: We have demonstrated that teeth engineered on biodegradable polymer scaffolds form odontoblast-like cells and dentin, have a pulp chamber, possess Hertwig’s root sheath epithelia, possess putative cementoblasts, have a morphologically correct enamel organ consisting of ameloblasts, stellate reticulum, stratum intermedium, and have fully formed dental enamel. Immunohistochemical analysis confirms the presence of collagen type I within the engineered dentin and the presence of amelogenin within the engineered enamel. These data provide solid evidence that dentin and enamel can be grown from dissociated porcine tooth tissues seeded onto biodegradable polymer scaffolds.