Failure to properly heal bone fractures is associated with significant morbidity and mortality. Additionally, the need to properly form bone is vital to successful outcomes from surgeries ranging from joint replacement to spinal fusion to dental implants. In an aging population, the biomedical need is even greater. The recent identification of skeletal progenitor cells, composed of 8 subpopulations, capable of forming all three components of the skeleton, bone, cartilage and stroma, promises to have a major impact on improving the outcomes for these therapies. A key question that remains is what are the factors affecting eventual cell fate of these skeletal progenitor cells. Our data on the matricellular protein DEL1 have shown it has an impact on skeletal repair. We have preliminary data indicating that it has a direct effect on skeletal progenitor cell biology and we propose that DEL1 plays an important role in their biology. Prior to this, our data had shown DEL1 had a biological effect on chondrocytes, but not on osteoblasts. We demonstrated decreased numbers of mouse skeletal stem cells (mSSCs), and bone, cartilage, stroma progenitors (BCSPs), the skeletal progenitor most important in fracture healing in the fracture callus, and we propose the mechanism for decreased bone formation after fracture is due to an effect on skeletal progenitors ability to expand after fracture. We plan on examining this first by isolating mSSCs and BCSPs, from knockout (KO) and wild type (WT) mice and comparing their biology. We will examine the biology of BCSPs using in vitro assays for cell proliferation, osteogenic and chondrogenic potential. BCSPs develop a different phenotype termed the fracture BCSP (f-BCSP) that has greater proliferative ability and osteogenic potential after fracture, and we will test whether the same change occurs in our KO mice. We will perform in vivo assays for osteogenesis by implanting BCSPs into the renal capsule to examine how well they form bone. A key question in understanding how DEL1 affects formation of bone after fracture is to understand what happens to skeletal progenitors after fracture in WT and KO mice. We will use lineage tracing techniques with mice currently in our laboratory to examine the fate of daughter cells from individual clones of skeletal progenitors cells. The final series of experiments represent our initial approaches to translating use of DEL1 into potential therapies. First, we will perform a carefully controlled microarray experiment to identify pathways altered in KO skeletal progenitors compared to WT. We will examine the ability of exogenous DEL1 to promote viability and growth of skeletal stem cells grown in vitro. We will test the ability of DEL1 to stimulate bone and cartilage formation of skeletal progenitor cells implanted into the renal capsule. Finally, we will examine whether exogenous DEL1 protein placed at a fracture site can enhance normal fracture healing. We will also use a separate model of poor fracture healing with mice that have undergone hindlimb irradiation. In summary, we will examine how Del1 affects the biology of skeletal progenitor cells. In addition to providing steps to potentially translate these discoveries into therapies, these experiments can provide us with mechanistic answers about how lack of Del1 leads to decreased bone in fracture healing. They will provide insight into the factors that affect the potential fate of skeletal progenitor cells.
The experiments proposed here are aimed at improving our understanding of skeletal stem cells. These are cells capable of producing all three components of the skeleton, bone, cartilage and stroma. Understanding the biology of these skeletal stem cells is highly relevant for the healthcare of veterans. The need for new bone formation is important in a number of surgical procedures including spinal fusion, joint replacement and dental implants. These surgeries represent some of the most common procedures performed in the veteran population. A major concern in the success of these procedures is the formation of quality new bone to support the secure the implant. Understanding how to improve bone formation can decrease complications of all these procedures. Furthermore, it has the potential to allow for new bone formation in critical defects of returning warfighters who are suffering large numbers of extremity injuries in current conflicts.
|Wang, Zhen; Tran, Misha C; Bhatia, Namrata J et al. (2016) Del1 Knockout Mice Developed More Severe Osteoarthritis Associated with Increased Susceptibility of Chondrocytes to Apoptosis. PLoS One 11:e0160684|