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

在重新提交我们的项目申请时，我们希望将最初审查时出现的建议纳入其中。我们已经完全修改了提案，并以颜色突出了最重要的改进，主要是参考审稿人的建议。特别是，我们澄清了重组蛋白的生产和他们的选择过程。进一步阐明了蛋白质在几丁质凝胶中的应用及其与多孔海绵样支架的加工和区别，以及该材料在骨再生方面的未来应用前景。正如建议的那样，我们已经把后一点放在了正确的位置，更多地强调了甲壳类蛋白质影响矿化的基础研究。6 .这导致广泛修订和更新工作计划的最新水平和重点。本文简要介绍了在大连理工大学和北京大学进行的补充初步实验。因此，我们相信，风险对于可预见的知识增益是合适的，正如在新增加的风险评估部分所讨论的那样。我们仍然着迷于甲壳类动物在换壳过程中的高矿化潜力，这优于哺乳动物的类似过程。我们的目标是依靠材料科学和生物专业知识的协同作用，实现这一潜力。初步的实验，体外和体内矿化过程的知识和我们现有的候选蛋白质管道正在加强这种方法。为了了解发生在甲壳类动物体内和体外条件下的矿化过程，我们将首先对蛋白质与几丁质基凝胶进行结合分析。随后，我们将在专门设计的矿化室中研究甲壳类双功能蛋白在特定条件下的矿化介导特性。由此产生的仿生材料的成骨诱导潜能将通过成骨细胞培养进行研究。由Amir Sagi教授、Thomas Hanke博士和Benjamin krupppke博士共同发起的BMBF项目建立了BGU和TUD的长期合作关系，我们将小龙虾的血淋巴蛋白固定在多孔支架上。跨学科联盟的附加价值在这里已经很明显了。在未来，作为小龙虾矿化外骨骼一部分的双功能小龙虾蛋白可能成为含有几丁质的仿生可注射骨替代品的有用组成部分，具有快速矿化特性，以支持骨再生。

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.