Mechanisms Underlying Heterotopic Ossification Heterotopic ossification (HO) is bona fide bone formation outside of the normal skeletal system. The precise mechanisms underlying HO are unknown, although numerous signaling pathways and cellular components, local and systemic, have been implicated. Our previous work demonstrated that dysregulation of the neuroinflammatory factor, substance P (SP), along with mast cells and macrophages, leads to elevated bone morphogenetic protein (BMP) signaling that triggers the pathological process. Glast and Tie2 expressing mesenchymal stem/progenitor cells (MSCs) respond to the inductive signals and form ectopic bone through endochondral ossification. Our central hypothesis is that unlike normal skeletogenesis, the process of HO is an injury/inflammation induced, abnormal local morphogenic phenomenon. Specifically, multiple disease specific contributing factors, including both cellular and extracellular molecular elements, co-ordinate with each other to form a unique hierarchical microenvironment, similar to endogenous stem cells niches, to induce and propagate HO. In the niche, MSCs recruited from local tissues are the cells of origin of HO, and specific local supportive cellular (e.g. local neres, mast cells) and molecular (e.g. SP) components are crucial to regulate the stereotyped self-renewal, proliferation, chondrogenic and osteogenic differentiation of these MSCs. Thus development of therapeutic approaches must consider the role of the local innervation and mast cells as well as local sources of cells of origin of HO. This requires an interdisciplinary approac to study the niche as a minimum functional unit, the goal of this proposal. Understanding the characteristics of the cells of origin of HO will be necessary for precise therapeutic targeting to prevent or limit the disorder. The first part of the proposed studies will examine the niche dwelling cells that directly participate in HO. The second part will study the niche supportive cells and key molecular components that initiate and propagate HO. A major focus will be to examine the therapeutic effectiveness in preventing or limiting HO by inhibiting factors, such as SP, that play a central role in the HO niche. Thus the overall goal is to develop a therapy for thi debilitating disorder for which there currently is no effective treatment.