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- Author or Editor: Mark E. Haskins x
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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 determine matrix metalloproteinase (MMP) activity in synovial fluid (SF) obtained from the joints of dogs with degenerative joint disease (DJD) secondary to various underlying conditions.
Sample Population—35 samples of SF obtained from 18 clinically normal (control) dogs and 34 samples of SF obtained from 17 dogs with DJD; dogs with DJD were from 2 populations (client-owned dogs and research dogs that had DJD secondary to the lysosomal storage disease mucopolysaccharidosis VII).
Procedure—MMP activity in samples of SF was semiquantitatively examined by use of gelatin or casein zymography. Western blot analysis was performed by use of antibodies for MMP-2 and MMP-9. In addition, in situ MMP activity was observed in sections of synovial membrane obtained from healthy and osteoarthritic joints.
Results—Samples of SF from osteoarthritic joints had higher MMP-2 activity and dramatically increased MMP-9 activity, compared with values for healthy joints. Substrate-overlay analyses indicated minimal gelatin-degrading activity in synoviocytes obtained from control dogs, whereas greater activity was seen in osteoarthritic synoviocytes, with additional activity in the underlying tissue.
Conclusions and Clinical Relevance—Higher MMP-2 activity and dramatic increases in MMP-9 activity were associated with the osteoarthritic state, even though MMP-2 activity was detected in healthy joints. This study expands information on MMP production in SF of osteoarthritic joints in other species and documents the similarity of MMP activity patterns regardless of the cause of DJD. (Am J Vet Res 2003;64:1225–1233)
