Gene Expression Profile During the Repair Process in a Rat Calvarial Bone Critical Size Defect

Abstract

Bone is a complex tissue, providing support and protection while also adapting to the body's mechanical needs. In response to injury or fracture, bone has a significant capability for self‐repair and regeneration that requires the regulated expression of diverse families of genes that coordinate complex interactions among various cell types. Studies on bone non‐union have led to the critical size defect (CSD) theory, which states that as bone defects increase in size, a point will be reached where bone union will not occur and the bone will not heal spontaneously during the lifetime of the animal. CSDs fail to heal and regenerate bone, while noncritical defects produce an acceptable repair after bone loss. To improve outcome following significant bone loss we sought to identify genes differentially expressed between an 8 mm (CSD) and 4 mm (non‐CSD) size defects in a rat calvarial bone defect model. Rat whole genome microarrays were used to measure gene expression over the course of bone healing, and analysis of differentially expressed genes (DEGs) with Cytoscape® software revealed multiple temporal patterns of fold differences for DEGs. The patterns indicated that greatest number of differences occur at the earliest time point post‐injury (day 1), and starting at day 7 and continuing through day 21. Gene ontology and functional classification of genes altered in expression between the CSD and non‐CSD was conducted using Ingenuity Pathway Analysis software®. During the earliest (day 1,3) and later (day 14,21) times post injury, multiple DEGs and gene networks related to cell death and survival, cancer, connective tissue development and function, DNA Replication/recombination/repair, skeletal and muscular system development and function, and others, were identified. At the early time points TGF‐β1 and VEGFα signaling pathways are down modulated in CSD relative to non‐CSD include. Pathways that regulate movement of lymphocytes and activation of antigen presenting cells, as well as NFk‐B signaling, are upregulated at later times post‐ injury in CSD compared to non‐CSD. We conclude that genes and signaling pathways that promote healing and tissue homeostasis are suppressed in the CSD, in favor of activation of pro‐inflammatory signaling.Disclaimer: Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.Support or Funding InformationThis work was supported by funding from the United States Army Medical Research and Material Command Combat Casualty Care and Clinical and Rehabilitative Medicine Research Programs