Abstract:

BACKGROUND: Classic isolation method of bone marrow mesenchymal stem cells (BMSCs) is Percoll density gradient centrifugation. Blood cell component was removed. However, this method is complicated. Preparation density was needed when isolating dog bone marrow. Moreover, centrifugation was frequent, which had a great damage to cells.

OBJECTIVE: To establish methods of the isolation, proliferation, culture and osteoinduction of canine BMSCs, and observe the in vitro proliferation and ability to osteoinduction differentiation.

METHODS: 10 mL bone marrow was extracted from dog posterior superior iliac spine, heparin anticoagulation, diluted using Hanks juice, treated with 1.077 g/mL Ficoll solution 3 mL, and centrifuged at 2 000 r/min for 20 minutes. Karyocytes were absorbed to form white cloudlike layering interface, and then centrifuged twice using DMEM supplemented with fetal bovine serum, incubated at 12×10⁴/cm² at 37 ℃ in a 5% CO₂ incubator. Following subculture, cells were incubated in DMEM containing dexamethasone, β-sodium phosphoglycerol and ascorbic acid 2-phosphate. Immunocytochemical staining and immunofluorescence staining were utilized to detect osteocalcin, osteopontin and type Ⅰ collagen expression in osteoblasts. Alkaline phosphatase staining and alizarin red staining were performed.

RESULTS AND CONCLUSION: 1.077 g/mL Ficoll density gradient centrifugation was used to isolate karyocytes that were significant compared with Percoll solution. Obtained BMSCs had high purity, good growth and the mean doubling time was 24 hours. Following in vitro osteogenic incubation of dog BMSCs, osteocalcin, osteopontin and type Ⅰ collagen showed positive expression. Alkaline phosphatase staining demonstrated bluish-green cytoplasm. Alizarin red staining showed red nodes in extracellular matrix, and could differentiate into osteoblasts in vitro.