Objective: To investigate the relationship between runt-related transcription factor 2 (RUNX2) expression in canine nucleus pulposus (NP) cells and intervertebral disk aging in chondrodystrophoid dogs. Animals: 7 healthy Beagles (mean age, 35.6 months) and 11 Dachshunds with herniated disks (mean age, 61 months). Procedures: All dogs underwent MRI examination of the thoracic and lumbar vertebral column immediately before sample collection under general anesthesia. The disk center–to–CSF T2-weighted signal intensity ratio was determined for healthy Beagles. Samples of NP were obtained from nonherniated disks in healthy Beagles and from herniated disks during surgical treatment of hospitalized Dachshunds. Samples were evaluated for RUNX2 and matrix metalloproteinase 13 transcript expression via reverse transcriptase PCR assay; RUNX2 protein expression was evaluated via immunohistochemical analysis, and correlation between these variables and age of dogs was evaluated. A 3′ and 5′ rapid amplification of cDNA ends method was used to identify the RUNX2 coding region. Results: RUNX2 cDNA had > 97% conservation with the human cDNA sequence and approximately 95% conservation with the mouse cDNA sequence; RUNX2 and matrix metalloproteinase 13 mRNA expression and RUNX2 protein expression in NP cells were positively correlated with age. The disk center–to–CSF T2-weighted signal intensity ratio was negatively correlated with RUNX2 protein expression in the NP of healthy dogs. Conclusions and Clinical Relevance: Results indicated that RUNX2 mRNA and protein expression in the NP are enhanced in aging intervertebral disks in dogs.

Objective: To isolate and characterize mesenchymal stem cells (MSCs) from canine muscle and periosteum and compare proliferative capacities of bone marrow-, adipose tissue-, muscle-, and periosteum-derived MSCs (BMSCs, AMSCs, MMSCs, and PMSCs, respectively). Sample: 7 canine cadavers. Procedures: MSCs were characterized on the basis of morphology, immunofluorescence of MSC-associated cell surface markers, and expression of pluripotency-associated transcription factors. Morphological and histochemical methods were used to evaluate differentiation of MSCs cultured in adipogenic, osteogenic, and chondrogenic media. Messenger ribonucleic acid expression of alkaline phosphatase, RUNX2, OSTERIX, and OSTEOPONTIN were evaluated as markers for osteogenic differentiation. Passage-1 MSCs were counted at 24, 48, 72, and 96 hours to determine tissue-specific MSC proliferative capacity. Mesenchymal stem cell yield per gram of tissue was calculated for confluent passage-1 MSCs. Results: Successful isolation of BMSCs, AMSCs, MMSCs, and PMSCs was determined on the basis of morphology; expression of CD44 and CD90; no expression of CD34 and CD45; mRNA expression of SOX2, OCT4, and NANOG; and adipogenic and osteogenic differentiation. Proliferative capacity was not significantly different among BMSCs, AMSCs, MMSCs, and PMSCs over a 4-day culture period. Periosteum provided a significantly higher MSC yield per gram of tissue once confluent in passage 1 (mean ± SD of 19,400,000 ± 12,800,000 of PMSCs/g of periosteum obtained in a mean ± SD of 13 ± 1.64 days). Conclusions and Clinical Relevance: Results indicated that canine muscle and periosteum may be sources of MSCs. Periosteum was a superior tissue source for MSC yield and may be useful in allogenic applications.