Project Summary/Abstract Large bone defects caused by trauma, infection, or cancer lead to thousands of amputees and millions of dollars in costs. Recently, a two-staged surgical regimen, Masquelet’s Induced Membrane Technique (MIMT), has presented a revolutionary way to reconstruct critical-sized defects that is more ideal than the current standards and could serve a wider patient population. More importantly, it presents a unique opportunity to discover new bone regeneration biology which could be applied to other bone formation situations (i.e. non- union, fusions) and possibly other tissues. The key feature of MIMT is an autologous foreign-body membrane that forms around an implanted bone cement spacer. Weeks to months later during a second surgery, the spacer is removed and the membrane compartment is filled with morselized bone graft. The technique can heal defects as large as 25 cm, more than triple the traditional maximum volume treatable via grafting without the membrane. However, MIMT’s regeneration mechanisms are completely unknown. All that is certain is that the membrane is necessary. Its critical functions have not yet been defined, so there is no evidence to improve its clinical application or harness the biological principles for other tissue regeneration scenarios. A critical barrier to investigating MIMT’s mechanisms is that it has only been thoroughly established in rats and larger animals. The number of genetic tools and reagents available for these species are far fewer than those available for mice. Thus, establishing a mouse-based MIMT model would be very powerful and allow much more in depth study and manipulation. To date, only one study has used a mouse for MIMT, but the second stage was not attempted and the first stage study design did not mirror well what has been done in other species. The goals of this proposal are to establish a mouse-based MIMT model, compare/contrast this model with what is known in other species, and use tools only available in mice to answer some fundamental MIMT questions. We hypothesize that a mouse model is feasible and that host cells, specifically osteoblasts, are mainly responsible for new bone generation. To test this hypothesis, we will first compare/contrast membrane formation and bone regeneration dynamics in normal C57BL/6J mice with what is already known from other species – most specifically rats. After establishing that a mouse MIMT model is feasible, we will use transgenic C57BL/6J mice to determine the contributions of host/graft cells to regeneration. Information gained from these experiments will be combined to target specific cells/genes/pathways in host/graft mouse tissues in a future R01.