IDENTIFICATION OF DIFFERENTIALLY EXPRESSED CRITICAL GENES IN CHONDROCLASTS AND OS

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DESCRIPTION (provided by applicant): During endochondral ossification, mineralized cartilage matrix is resorbed prior to bone formation and remodeling. While this cartilage matrix resorption is mediated by cells that exhibit many phenotypic features of osteoclasts, a longstanding question remains as to whether differences exist in the gene expression between these chondroclasts and the better characterized bone resorbing osteoclasts. To address this question we will use our mouse femoral fracture model, in which calcified cartilage matrices in the early callus will be remodeled prior to the remodeling of bony callus, during late stages of healing. While histological evidence has shown distinct identities of chondroclasts and osteoclasts, little information is available regarding the molecular characteristics that differentiate their respective functions. Our goal is to compare the gene expression profiles of TRAP-positive cells resorbing mineralized cartilage matrix (chondroclasts) with that of cells resorbing bone matrix (osteoclasts). We will further determine which of these differentially expressed genes is important for the respective functions of chondrocytes versus osteoclasts. These studies will be performed on cells that are removed from histologic samples of fractured mouse bones. We believe that the matrix and environment surrounding chondroclasts and osteoclasts critically influences the expression profiles of these cells, which almost certainly derive from a common progenitor. Therefore, we have designed our studies to examine differences in gene expression in cells that are removed directly from their in vivo environment so that we can best appreciate differences in their gene expression profiles. Our central hypothesis posits that osteoclasts and chondroclasts have distinct patterns of gene expression, which facilitate their unique functions. We propose the following two Specific Aims to test this hypothesis: Specific Aim1: To examine the molecular differences between laser-captured TRAP-positive cells resorbing cartilage or bone matrices during fracture healing in mice. We hypothesize that TRAP-positive cells resorbing calcified cartilage matrix will exhibit a different gene expression profile than TRAP-positive cells resorbing bone matrix. Specific Aim2: To establish functional differences between chondroclasts and osteoclasts isolated from fractured femurs. We hypothesize that functional differences exist between osteoclast and chondroclasts, which facilitate matrix-specific resorption. Our proposed studies will for the first time demonstrate, using a fracture healing mouse model, whether there are functional differences in the molecular mechanisms regulating cartilage versus bone remodeling during endochondral ossification post-natally. PUBLIC HEALTH RELEVANCE: Skeletal development and fracture repair of long bones require successive cellular events that not only involve cartilage and bone cells, but also cells that remodel cartilage (chondroclasts) and bone (osteoclasts) matrices. Here we propose to identify molecular differences between these two cell types and further examine the critical genes that are required for chondroclasts to mediate cartilage matrix remodeling for proper fracture healing. Clinical evidence is emerging demonstrating the importance of cartilage matrix remodeling for proper skeletal development and normal fracture healing. Data obtained from these studies may help develop novel therapeutic modalities to accelerate fracture repair in young and aged populations.