Method: Mouse amelogenin gene was cloned into E. coli expression vector pDEST17 and mammalian expression vector pCMV6, respectively. Dental pulp (DP) and periodontal ligament (PDL) stem/progenitor cells were isolated per our prior methods. E.coli expressed recombinant mouse amelogenin (rM179) was purified using Cobalt-Sepharose6-resin affinity column. Wild-type DP and PDL cells were transfected with pCMV6-amelogenin vector and then subjected to G418 selection to obtain an exclusive population of amelogenin-expressing cells. The expression of amelogenin was confirmed with western-blot. Differentiation was examined using ALP/von Kossa staining and real-time qPCR. In a separate experiment, DP or PDL cells were seeded in calcium phosphate scaffolds and implanted subcutaneously in the SCID mice. The tissue formation was analyzed by histology, histomorphometry and immunohistochemistry.
Result: 1) Recombinant amelogenin showed robust biological activity and induced the odontoblastic/osteogenic differentiation of PDL and DP cells as illustrated by calcified matrix deposition. Consistently, DSPP, DMP-1 were upregulated in DP cells, whereas CEMP-1 was up-regulated in PDL cells; 2) Compared to vector controls, DP and PDL cells constitutively expressing amelogenin (DP-AML, PDL-AML) showed enhanced calcified matrix deposition as well as increased marker gene expression; 3) The DP-AML cells became polarized and laid down DSPP positive matrix on the surface of calcium phosphate scaffold in vivo. The PDL-AML cells formed a cementum-like structure with cementum markers including CEMP-1 and CAP, in addition to putative Sharpey’s fibers.
Conclusion: Amelogenin promotes dentinogenesis and cementogenesis both in tissue culture and in vivo. Additional large animal and human studies are warranted to investigate whether amelogenin orchestrates dentin and cementum regeneration.
Keywords: Amelogenin, Dentin and Regeneration
See more of: Pulp Biology & Regeneration Research