Evaluation of bioengineered osteodentin-like tissue formation on silk scaffolds

Objectives: To improve upon methods for bioengineered mineralized dental tissue formation, we examined the utility of silk scaffolds, based on their successful use in bone tissue engineering. Methods: Four types of hexafluoroisopropanol (HFIP) silk scaffolds - 250 and 550 micron diameter pores, with or without incorporated RGD peptide - were seeded with cultured 4 day post natal (dpn) rat tooth bud cells and implanted to rat omenta for 20 weeks. The implants were harvested and analyzed using radiographic, histological, and immunohistochemical methods. Results: Bioengineered mineralized osteodentin-like tissue formation was observed in all cell-seeded scaffolds, and not in unseeded control scaffolds. Epithelial cell differentiation, and enamel formation, was not observed in any of the implants. Mineralized tissue formation was most robust in 550 micron pore sized silk scaffolds with RGD, and least robust in 250 micron pore size scaffolds without RGD. Bioengineered osteodentin formation appeared to be guided by the size and shape of the silk scaffold pores, as revealed by polarized light imaging of collagen fiber alignment along the scaffold surfaces. Immunohistochemical analysis of AM, BSP, DSPP, OCN, and OPN expression was used to further characterize osteodentin-like tissues. Conclusions: These results are the first to demonstrate the utility of silk scaffolds for mineralized dental tissue engineering applications, and suggest that large pore sized silk scaffolds can be used to fabricate osteodentin of specified size and shape. This work was supported by NIH/NIDCR grant DE016132 (PCY), and NIH Center Number: P41 EB002520 (DLK).