Interaction of Age and Mechanical Stability on Bone Defect Healing: An Early Transcriptional Analysis of Fracture Hematoma in Rat

Among other stressors, age and mechanical constraints significantly influence regeneration cascades in bone healing. Here, our aim was to identify genes and, through their functional annotation, related biological processes that are influenced by an interaction between the effects of mechanical fixation stability and age. Therefore, at day three post-osteotomy, chip-based whole-genome gene expression analyses of fracture hematoma tissue were performed for four groups of Sprague-Dawley rats with a 1.5-mm osteotomy gap in the femora with varying age (12 vs. 52 weeks - biologically challenging) and external fixator stiffness (mechanically challenging). From 31099 analysed genes, 1103 genes were differentially expressed between the six possible combinations of the four groups and from those 144 genes were identified as statistically significantly influenced by the interaction between age and fixation stability. Functional annotation of these differentially expressed genes revealed an association with extracellular space, cell migration or vasculature development. The chip-based whole-genome gene expression data was validated by q-RT-PCR at days three and seven post-osteotomy for MMP-9 and MMP-13, members of the mechanosensitive matrix metalloproteinase family and key players in cell migration and angiogenesis. Furthermore, we observed an interaction of age and mechanical stimuli in vitro on cell migration of mesenchymal stromal cells. These cells are a subpopulation of the fracture hematoma and are known to be key players in bone regeneration. In summary, these data correspond to and might explain our previously described biomechanical healing outcome after six weeks in response to fixation stiffness variation. In conclusion, our data highlight the importance of analysing the influence of risk factors of fracture healing (e.g. advanced age, suboptimal fixator stability) in combination rather than alone.

[1]  M. Delp,et al.  Decreases in Bone Blood Flow and Bone Material Properties in Aging Fischer-344 Rats , 2002, Clinical orthopaedics and related research.

[2]  G. Duda,et al.  Mesenchymal Stem Cells Regulate Angiogenesis According to Their Mechanical Environment , 2007, Stem cells.

[3]  G. Duda,et al.  A new device to control mechanical environment in bone defect healing in rats. , 2008, Journal of biomechanics.

[4]  D. Hu,et al.  Molecular aspects of healing in stabilized and non‐stabilized fractures , 2001, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[5]  S. Skak,et al.  Femoral shaft fracture in 265 children. Log-normal correlation with age of speed of healing. , 1988, Acta orthopaedica Scandinavica.

[6]  R. Meyer,et al.  Gene Expression in Older Rats with Delayed Union of Femoral Fractures , 2003, The Journal of bone and joint surgery. American volume.

[7]  Yu-Chen Huang,et al.  Mechanical Strain Induces Collagenase-3 (MMP-13) Expression in MC3T3-E1 Osteoblastic Cells* , 2004, Journal of Biological Chemistry.

[8]  T A Einhorn,et al.  The cell and molecular biology of fracture healing. , 1998, Clinical orthopaedics and related research.

[9]  I. Owan,et al.  Aging changes mechanical loading thresholds for bone formation in rats , 1995, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[10]  Jill A. Helms,et al.  Altered fracture repair in the absence of MMP9 , 2003, Development.

[11]  Matko Bosnjak,et al.  REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms , 2011, PloS one.

[12]  M. Takigawa,et al.  Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1. , 1999, Journal of biochemistry.

[13]  Thomas A Einhorn,et al.  Fracture healing as a post‐natal developmental process: Molecular, spatial, and temporal aspects of its regulation , 2003, Journal of cellular biochemistry.

[14]  G. Duda,et al.  Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: involvement of VEGF mechano-regulation. , 2010, Bone.

[15]  Georg N Duda,et al.  Insight into the molecular pathophysiology of delayed bone healing in a sheep model. , 2010, Tissue engineering. Part A.

[16]  D. Hutmacher,et al.  CD73 and CD29 concurrently mediate the mechanically induced decrease of migratory capacity of mesenchymal stromal cells. , 2011, European cells & materials.

[17]  E. Vuorio,et al.  Expression of Cathepsins B, H, K, L, and S and Matrix Metalloproteinases 9 and 13 During Chondrocyte Hypertrophy and Endochondral Ossification in Mouse Fracture Callus , 2000, Calcified Tissue International.

[18]  Z. Werb,et al.  How matrix metalloproteinases regulate cell behavior. , 2001, Annual review of cell and developmental biology.

[19]  G. Duda,et al.  Influence of age and mechanical stability on bone defect healing: age reverses mechanical effects. , 2008, Bone.

[20]  R. Carano,et al.  Angiogenesis and bone repair. , 2003, Drug discovery today.

[21]  C. Rubin,et al.  Suppression of the osteogenic response in the aging skeleton , 1992, Calcified Tissue International.

[22]  H. Bail,et al.  An easily reproducible and biomechanically standardized model to investigate bone healing in rats, using external fixation / Ein leicht reproduzierbares und biomechanisch standardisiertes Modell zur Untersuchung der Knochenheilung in der Ratte unter Verwendung eines Fixateur Externe , 2007, Biomedizinische Technik. Biomedical engineering.

[23]  M. Heller,et al.  The initial phase of fracture healing is specifically sensitive to mechanical conditions , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[24]  G. Duda,et al.  Insights into Mesenchymal Stem Cell Aging: Involvement of Antioxidant Defense and Actin Cytoskeleton , 2009, Stem cells.

[25]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[26]  D. Hutmacher,et al.  Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing: is osteoclast activity the key to age-related impaired healing? , 2010, Bone.

[27]  Z. Werb,et al.  Altered endochondral bone development in matrix metalloproteinase 13-deficient mice , 2004, Development.

[28]  P. Megas,et al.  Classification of non-union. , 2005, Injury.

[29]  L. Claes,et al.  Early dynamization by reduced fixation stiffness does not improve fracture healing in a rat femoral osteotomy model , 2009, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[30]  A. M. Phillips.  Overview of the fracture healing cascade. , 2005, Injury.

[31]  D. M. Banks,et al.  Age and ovariectomy impair both the normalization of mechanical properties and the accretion of mineral by the fracture callus in rats , 2001, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[32]  R. Hayda,et al.  Pathophysiology of delayed healing. , 1998, Clinical orthopaedics and related research.

[33]  Alex E. Lash,et al.  Gene Expression Omnibus: NCBI gene expression and hybridization array data repository , 2002, Nucleic Acids Res..

[34]  Z. Werb,et al.  How Proteases Regulate Bone Morphogenesis , 2003, Annals of the New York Academy of Sciences.

[35]  D Kaspar,et al.  Effects of Mechanical Factors on the Fracture Healing Process , 1998, Clinical orthopaedics and related research.

[36]  T A Einhorn,et al.  Growth Factor Regulation of Fracture Repair , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[37]  Eleftherios Tsiridis,et al.  Current concepts of molecular aspects of bone healing. , 2005, Injury.