Crustacean bifunctional proteins in mineralized exoskeleton – a model for biomimetic injectable bone substitutes

• Applicant: Dr.-Ing. Benjamin Kruppke
• Subject Area: Biomaterials
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 538923079

With this resubmission of our project application, we would like to incorporate the suggestions that appeared at the initial review. We have completely revised the proposal and have highlighted in color the most significant improvements that primarily refer to the reviewers' advice. In particular, we have clarified the production of the recombinant proteins and their selection process. The use of proteins in chitin gels and their processing and distinction from porous sponge-like scaffolds were further clarified, as well as the perspective of the material for a future application in the context of bone regeneration. As suggested we have placed the latter point into perspective adding more emphasis on the basic research of mineralization influenced by crustacean proteins. 0This leads to an extensive revision and update of the state of the art and focus of the work plan. Complementary preliminary experiments performed at TUD and BGU are briefly explained and referred to. Thus, we believe, the risk is appropriate to the foreseeable gain in knowledge, as discussed in the newly added risk assessment section. We are still fascinated by the high mineralization potential of crustaceans during molting, which is superior to similar processes in mammals. We aim to make this potential accessible relying on the synergy of our materials science and biological expertise. The preliminary experiments, knowledge of in vitro and in vivo mineralization processes and our existing pipeline of protein candidates are enforcing the approach. To understand the mineralization processes occurring in crustaceans and under in vitro conditions, we will first perform binding analyses of the proteins to chitin-based gels. Subsequently, we will investigate the mineralization-mediating properties of the crustacean bifunctional proteins under defined conditions in a specially designed mineralization chamber. The osteoinductive potential of the resulting biomimetic material will be investigated using osteoblast cell cultures. The long-standing cooperation between BGU and TUD was established by Prof. Amir Sagi, Dr. Thomas Hanke, and Dr. Benjamin Kruppke based on a joint BMBF project in which we immobilized hemolymphatic proteins from crayfish on porous scaffolds. The added value of the interdisciplinary consortium was already evident here. In the future, bifunctional crayfish proteins found as part of the crayfish mineralized exoskeleton could be useful components of a chitin-containing biomimetic injectable bone substitute with rapid mineralization properties to support bone regeneration.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professor Dr. Amir Sagi, Ph.D.