D. M. Struck1, T. V. Boyko1, R. K. Mann4, M. T. Longaker4, C. K. Chan4, G. Yang1,2,3 1Stanford University,Department Of Surgery,Palo Alto, CA, USA 2University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 3Birmingham VA Medical Center,Birmingham, ALABAMA, USA 4Stanford University,Stanford University School Of Medicine,Palo Alto, CA, USA
Introduction: Of the millions of people who suffer a bone fracture every year, 5-10% will have delayed or impaired healing leading to major economic and quality of life burdens. We have shown the developmentally-regulated endothelial cell locus 1 protein (DEL-1) acts as a growth factor for mouse Bone Cartilage Stroma Progenitors (BCSPs), the skeletal progenitor population most important in fracture healing. DEL-1 is a modular protein with three EGF like repeats, two discoidin domains and an RGD motif. Deletion of Del-1 leads to mice that heal fractures with decreased bone. Based on this phenotype and the presence of an RGD motif, we hypothesized that DEL-1 interacts with integrins at this binding site to promote BCSP proliferation to the fracture callus during bone healing.
Methods: Polymerase chain reaction mutagenesis was used to create a mutant Del-1 DNA construct where the RGD (Arg-Gly-Asp) sequence was substituted with RAD (Arg-Ala-Asp). Both DEL-1 wildtype (WT) and DEL-1 RAD mutant (Mut) was produced using a baculovirus expression system, and purified with immobilized metal affinity and size-exclusion chromatography. Del-1 knockout mice underwent mid-femoral fractures with internal fixation followed by local application of hydrogels loaded with DEL-1 WT, DEL-1 Mut or blank controls. Additional controls were wild type mice treated with blank hydrogel. BCSPs were isolated from the fracture callus 7 days after fracture using FACS. Untreated Del-1 knockout BCSPS were also plated on 24 well plates coated with DEL-1 WT, DEL-1 RAD Mutant or BSA control. Proliferation was measured every day for a week.
Results: The addition of DEL-1 WT to Del-1 knockout mice resulted in increased callus formation compared to control (p<0.05). This was correlated with increased BCSPs at the fracture callus (p< 0.05). In vitro, the addition of DEL-1 WT led to increased proliferation of BCSPs (p< 0.05). In contrast, the addition of DEL-1 Mut had no effect on either the size of the fracture callus or the number of BCSPs at the fracture site. In vitro, there was no impact on proliferation of DEL-1 Mut on BCSPs.
Conclusion: DEL-1 WT rescued the phenotype of poor fracture healing in the Del-1 knockout mice leading to increased fracture callus size with increased numbers of BCSPs. This was correlated with stimulation of proliferation in vitro. In contrast, the DEL-1 Mut had no impact on fracture callus size, mobilization of BCSPs or proliferation of BSCPs. Based on the known function of the RGD motif, we conclude that DEL-1 affects BCSPs through binding of integrins using the RGD motif.