gms | German Medical Science

Objectives: The healing of skeletal fractures involves a cascade of overlapping cellular events. This study aims to deepen the understanding of molecular networks orchestrating these events.

Methods: Standard mid diaphyseal closed fracture in the left femur of male 8-10 weeks old C57BL/6N mice were analyzed at (day = D) D3, D7, D10, D14, D21 & D28 post fracture (N = 5 / time point). Total RNA was prepared for whole genome expression profiling using Illumina µ array kit. Data normalization and analysis was performed using the "R" platform. The thresholds for filtering the differentially expressed genes (DEG) were set at FC (fold-change) ≥ |1| and p-value ≤ 0.01. Functional enrichment analysis was performed using NCBI-DAVID to identify genes of immune system response, mitochondria, ribosome, angiogenesis, ossification and extracellular matrix (ECM). Cytoscape was used to perform the regulatory network analysis between these biological events.

Results and Conclusion: Despite the level of understanding behind the fracture healing process, very little is known about the complexity of overlapping genes during different stages of healing. This study addresses the differentially expressed (DE) overlapping genes in the above mentioned biological processes. After gene ontology analysis, 35 genes were DE in more than one biological processes during the healing process. These genes were further analyzed based on their correlation with the cellular events. As an overlapping gene between immune response, mitochondrion and angiogenesis: Glutathione Peroxidase 1 (Gpx1) was DE at the early and later stages of healing to support the reactive oxygen species homeostasis. Similarly, the correlated gene between ossification and ribosome: Ubiquitin B (Ubb) was DE during the early and later stages to control the osteoblast differentiation. However, the angiogenesis, ECM and ossification co-related gene: Matrix metallopeptidase 2 (Mmp2) was significantly expressed until the endochondral ossification phase of the healing which plays a role in the remodeling of the vasculature as well as encodes the binding of collagen.

Currently, the detailed and regulatory role of these genes, the pathways and their involvement in different skeletal disorders are being investigated. However, the results indicate promising understanding of overlapping gene function between the cellular events.

Bone diseases or fractures leading to delayed or nun-union healing are often treated to enhance bone formation. Therefore, investigating gene expression overlapping -both chronologically and biologically- is crucial for the design of systemic or local therapeutic agents.