Mechanisms regulating bone healing and angiogenesis in osteonecrosis - Project Summary Osteonecrosis (ON) of the femoral head (ONFH) is one of the most severe hip diseases affecting all ages with an estimated 20+ million affected people worldwide. ONFH has become a major cause of total hip arthroplasty resulting in great financial burdens. Currently, multiple high prevalence risk factors have been identified including trauma, glucocorticoid use, alcohol abuse and genetics, but none of these are certain and the etiology of ON is unclear. There are no gold standards for the treatment of ON. Most treatment are focused on delaying disease progression to preserve the joint using surgical techniques. No effective biological treatment has been identified due to that little is known about the molecular mechanisms associated with ON healing. ON is caused by a disruption of blood supply leading to necrotic bone cell death. An imbalanced osteogenesis and adipogenesis with impaired angiogenesis is often presented in the process of ON healing. It is thus critical to fully characterize the process of stem cell differentiation to repopulate necrotic bone and the mechanisms regulating revascularization in necrotic bone healing. Towards this end, we have successfully established a rodent model showing consistent human ON phenotype with ischemic tissue damage to the whole joint. Using non-biased scRNA sequencing, we showed that Osterix positive (OSX+) bone progenitors are significantly expanded in ON compared to other progenitor populations. Interestingly, these OSX+ cells demonstrate an expanded and sporadic marrow distribution that does not associate with vessels. Moreover, OSX+ cells in ON contain an increased progenitor subpopulation that co-expresses PPARγ which is absent in control. We also determined that enhanced adipogenesis in ON is accompanied by elevated LDL and oxidative stress resulting in increased levels of oxidized low-density lipoprotein (oxLDL). Importantly, using both in vivo and in vitro assays we show that oxLDL impairs angiogenesis by downregulating endothelial Notch signaling. Our long-term goal is to uncover the key mechanisms in ON healing, and thus provide a molecular basis for future biological treatment of ON. Our central hypothesis is that OSX+ cells are the critical cell population responsible for imbalanced osteogenesis and adipogenesis observed in ON, which negatively affects the revascularization of necrotic bone via an oxLDL-Notch signaling mediated mechanism. We will attest our hypothesis through two highly-related but independent specific aims: Aim 1: To identify and define the OSX+ population of progenitors that repopulate necrotic bone marrow in ON; and Aim 2: To determine the oxLDL-Notch mediated signaling mechanisms that regulate angiogenesis in ON. We will investigate the above listed aims by using single cell RNA sequencing technique, lineage tracing, metabolomics and Notch loss-of-function and gain-of-function models of genetically engineered mouse. Successful completion of this project will advance our understanding and identify key mechanisms that regulate bone progenitor cells differentiation and angiogenesis in ON. We believe that this work will lay the foundation for future investigation of biological treatment in ON.
