Models for evaluating recombinant human ameloblastin role in periodontal regeneration

Porcine enamel matrix protein derivative (EMD: Emdogain®) is widely used to facilitate periodontal regeneration. Emdogain® was also found to be osteopromotive in long bone and in intermembranous cranial bone defects. Recently we've found that Emdogain® which is known to be composed mainly of amelogenin peptides, also contains several ameloblastin peptides. Ameloblastin was reported to be constantly expressed by ameloblasts from very early stages of amelogenesis. It is also transiently expressed by preodontoblasts in dentinogenesis and by cells of the herthwig's epithelial root sheath during root formation and cementogenesis. Using immunohistochemestry, we've identified the presence of ameloblastin also in rat mature cementum and alveolar bone. Objective: to test possible involvement of ameloblastin in regenerating processes, induced by Emdogain®. Results: two recombinant human ameloblastin proteins were produced using the baculovirus expression system. The first protein was expressed in the cytoplasm of insect cells (SF9), purified and characterized by SDS PAGE, Western blot and ESI/MS/MS sequencing, confirming the production of recombinant human ameloblastin protein. Another ameloblastin protein was produced and secreted into the medium, by SF9 insect cells. This protein was also characterized by SDS PAGE, Western blot. Methods: Two models will be employed to check the possible regenerating activity of the recombinant human ameloblastin. The first model is of 4mm circular parietal bone defects in rat calvarias that will be treated with the recombinant human ameloblastin protein in PGA carrier, with and without the use of a resorbable membrane. The second model is the periodontal window wound in rat mandibles which will be treated with the recombinant human ameloblastin protein in PGA carrier. Both models will be analyzed by µCT, histology, immunohistochemistry and in-situ hybridization. Conclusion: these models might indicate the involvement of ameloblastin in bone regenerating processes.

Work was supported by E.U. Framework5 "MATRIX" grant no:QLK3-CT-2001-00090.