BMP-2 mRNA expression after endothelial progenitor cell therapy for fracture healing.
OBJECTIVE
Endothelial progenitor cells (EPCs) represent a population of novel precursor cells with known ability to participate in angiogenesis. Our previous studies have shown that local EPC therapy significantly increased angiogenesis and osteogenesis to promote fracture healing in an animal bone defect model. However, the cellular and molecular mechanisms by which EPC therapy promotes fracture healing remain largely unknown. The purpose of this study was to quantify local bone morphogenetic protein (BMP-2) expression after EPC therapy for a rat segmental bone defect, in hopes of further defining the potential mechanisms by which EPCs promote fracture healing.


METHODS
EPCs were isolated from the bone marrow of syngeneic rats and cultured ex vivo for 7-10 days before transfer to the bone defect. A total of 56 rats were studied. The treatment group received 1 × 10 EPCs on a gelfoam scaffold at the bone defect, and control animals received gelfoam/saline only. Before euthanasia, radiographs of the femur were performed. Animals were euthanized at 1, 2, 3, and 10 weeks, and specimens from the fracture gap area were collected, pulverized, and total messenger RNA (mRNA) was extracted. BMP-2 mRNA was measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. All measurements were performed in triplicate.


RESULTS
All EPC-treated bone defects healed radiographically by 10 weeks, whereas control-treated defects developed a nonunion. The expression of BMP-2 mRNA was significantly elevated in EPC-treated defects relative to controls at week 1 (EPC, 0.59 ± 0.10; control, 0.31 ± 0.08; P = 0.05), week 2 (EPC, 0.40 ± 0.06; control, 0.23 ± 0.04; P = 0.04), and week 3 (EPC, 0.33 ± 0.06; control, 0.18 ± 0.03; P = 0.04), but not at week 10 (EPC, 0.31 ± 0.06; control, 0.21 ± 0.04, P = 0.15). The highest mean expression of BMP-2 in EPC-treated defects was observed at 1 week, with a progressive decline in BMP-2 expression noted thereafter.


CONCLUSIONS
These findings demonstrate that EPC-treated bone defects demonstrate both radiographic healing and elevated expression of BMP-2 relative to control-treated defects. These results provide further insight into the potential mechanisms by which EPC therapy may promote fracture healing and provide further evidence to suggest that the trophic actions of EPC therapy may be a critical factor in their contribution to fracture healing.
*Department of Surgery, Division of Orthopaedic Surgery, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada; and †Department of Surgery, Toronto Western Hospital, UHN, University of Toronto, Toronto, Ontario, Canada.
Ru
R
Li
Aaron
A
Nauth
Rajiv
R
Gandhi
Khalid
K
Syed
Emil H
EH
Schemitsch
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000096:biosynthesis*; D064987:Cell- and Tissue-Based Therapy; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D005264:Femoral Fractures / Q000503:physiopathology / Q000628:therapy*; D005269:Femur / Q000503:physiopathology; D017102:Fracture Healing / Q000502:physiology*; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000378:metabolism*; D054457:Tissue Scaffolds
D055396:Bone Morphogenetic Protein 2; D012333:RNA, Messenger