Effects of osteogenic inducers on cultures of canine mesenchymal stem cells.
OBJECTIVE
To examine age-related efficacy of bone morphogenetic protein (BMP)-2, ascorbate, and dexamethasone as osteogenic inducers in canine marrow-derived stromal cells (MSCs).


METHODS
Samples of femoral bone marrow obtained from 15 skeletally immature (< 1 year old) and 4 skeletally mature (> 1.5 years old) dogs.


METHODS
First-passage canine MSC cultures were treated with 100 microg of ascorbate phosphate/mL, 10(-7)M dexamethasone, 100 ng of BMP-2/mL, or a combination of these osteoinducers. On day 6, cultures were harvested for quantitation of alkaline phosphatase (ALP) activity and isolation of RNA to prepare cDNA for real-time polymerase chain reaction analyses of osteoblast markers.


RESULTS
Early markers of osteogenesis were induced in canine MSCs by BMP-2 but not dexamethasone. In young dogs, the combination of BMP-2 and ascorbate yielded the highest ALP mRNA concentrations and activity. This combination also induced significant increases in mRNA for osteopontin and runt-domain transcription factor 2. In comparison to MSCs from immature dogs, those from mature dogs had diminished ALP activity in response to BMP and ascorbate. Results for cultures treated with 3,4-dehydroproline suggested that ascorbate-induced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs.


CONCLUSIONS
BMP-2 was capable of inducing markers of osteogenesis in short-term cultures of canine MSCs. In MSCs obtained from skeletally immature dogs, ascorbate was required for maximal effects of BMP These results define optimal conditions for stem cell osteogenesis in dogs and will facilitate development of stem cell-based treatments for dogs with fractures.
Volk
Susan W
SW
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Diefenderfer
David L
DL
Christopher
Scott A
SA
Haskins
Mark E
ME
Leboy
Phoebe S
PS
D000367:Age Factors; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology*; D004285:Dogs / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000187:drug effects* / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D016212:Transforming Growth Factor beta; C011669:ascorbate-2-phosphate; D003907:Dexamethasone; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid