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Effects of osteogenic inducers on cultures of canine mesenchymal stem cells

Susan W. VolkDepartment of Clinical Studies, University of Pennsylvania, Philadelphia, PA 19104.

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David L. DiefenderferDepartment of Clinical Studies, University of Pennsylvania, Philadelphia, PA 19104.

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Scott A. ChristopherSchool of Veterinary Medicine, and the Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Mark E. HaskinsDepartment of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104.

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Phoebe S. LeboySchool of Veterinary Medicine, and the Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Abstract

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).

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

Procedure—First-passage canine MSC cultures were treated with 100 µg of ascorbate phosphate/mL, 10–7M 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 ascorbateinduced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs.

Conclusions and Clinical Relevance—BMP-2 was capable of inducing markers of osteogenesis in shortterm 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. (Am J Vet Res 2005;66:1729–1737)

Abstract

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).

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

Procedure—First-passage canine MSC cultures were treated with 100 µg of ascorbate phosphate/mL, 10–7M 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 ascorbateinduced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs.

Conclusions and Clinical Relevance—BMP-2 was capable of inducing markers of osteogenesis in shortterm 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. (Am J Vet Res 2005;66:1729–1737)