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Friday, Mar 22, 2013, 8:00 AM - 5:30 PM
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Notch Signaling in Mesenchymal Stem Cells (MSCs) and Tibial Fracture Callus Harvested from Geriatric Mice
+Geriatric Fractures (Trauma)
Nicole S. Belkin
, MD, Philadelphia, Pennsylvania
Lorraine L. Mutyaba
, BS, Philadelphia, Pennsylvania
Allison E. Williams
, Philadelphia, Pennsylvania
Lee McDaniel JR
, MS, Philadelphia, Pennsylvania
Derek L. Dopkin
, BA, Philadelphia, Pennsylvania
Kurt D. Hankenson
, DVM, Philadelphia, Pennsylvania
, MD, PhD, Philadelphia, Pennsylvania
INTRODUCTION: Morbidity associated with geriatric fractures can be attributed, in part, to compromised mesenchymal stem cell (MSC) function within the fracture callus and late onset of endochrondal ossification. The Notch signaling pathway has been shown to be important for healing of non-skeletal tissues in an age-dependent manner.
is to characterize age-based differences in Notch signaling in a mouse fracture healing model. This characterization has the potential to elucidate Notch-based clinical therapies for bone.
METHODS: Five-month-old C57BL/6 mice are reproductively and skeletally mature. Twenty-five-month old C57BL/6 mice represent the age of 25% survival and hence are “geriatric” mice. Five- and 25-month-old C57BL/6 mice underwent bilateral closed, transverse fractures of the tibial shaft with intramedullary pin fixation (IACUC approved). All mice were obtained from colonies maintained at the National Institute of Aging. At 0 days post-fracture (DPF), 10 DPF and 20 DPF tibial fracture calluses and MSCs were harvested. RNA was harvested from cell cultures and homogenized callus and reverse transcribed. Expression of Notch ligands, receptors and target genes were determined quantitatively using real-time PCR with Power SYBR green. One manufacturer's microarray chip was used to assess global changes in gene expression. Inter-array microarray data sets were normalized with R 2.15 and Bioconductor 2.1 using the RMA (robust multichip average) function of bioconductor with default parameters. RMA performs quantile normalization followed by median polish. Normalized data sets were loaded into MultiExperimentViewer 4.8, which executed a two factor ANOVA test with a p-value of 1%. The top 200 p-valued hits for age, healing and interaction significant genes, respectively, were loaded into DAVID. DAVID was used to scan for gene ontology and kegg term saturations with a p-value of 1%.
RESULTS: MSC from geriatric mice showed reduced basal expression of notch targets, Hey 1, Hey L and Hey 2, but showed responsiveness to ligand stimulation with Jagged-1. Microarray analysis of fracture callus showed differential expression in multiple notch signaling pathway genes (Fig.1.) There was upregulation in Notch receptors and target genes, consistent with our previous findings. Differences with age included increased expression of the Dll4 ligand, and down regulation of the Notch co-repressor, CIR.
DISCUSSION: An understanding of molecular differences between young and geriatric fracture healing has the potential to provide a basis for rational therapeutic targeting of specific pathways of fractures in the elderly.
MSC from geriatric mice exhibit reduced proliferation and adipogenesis, inconsistent osteogenesis and decreased basal notch activity. Of note, it has been shown that these geriatric MSC are responsive to Jagged-1 stimulation. Our collective data indicating differential activity of the Notch pathway in geriatric MSC and fracture callus derived cells would suggest that therapeutic targeting of notch signaling may be useful to improve geriatric fracture healing.
SIGNIFICANCE: This research aims to inform clinical therapies for bone repair by studying underlying differences at the gene expression level in fracture healing between young and aged mice that result in differences in biological healing.
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