Objective: To modify the digestion and culture of human osteoblasts for releasing a large amount of cells.
Methods: Bone tissue was harvested and digested by trypsin for several times to remove blood cells and fibroblasts. After cultured for a couple of days in incubator, bone chips were digested again by trypsin for several times, and a large amount of pure osteoblasts were released and used for researches. The remaining chips were digested by trypsin repeatedly until enough cells were harvested. Black nodes were seen in confluent cell layer. Osteoblasts were confirmed by their unique secretion of a large amount of alkaline phosphatase (AKP), osteocalcin (OCN) and type I collagen.
Results: Under fluorescent microscope, black node was "golden yellow" in color, a characteristic of bone node. Secretion of AKP (2.76 +/- 0.56) IU/ml and OCN (1.875 +/- 0.549) ng/ml of osteoblasts was higher than those of fibroblasts. There was only type I collagen in supernatant without type III.
Conclusion: The modified osteoblast digestion and culture can release a large amount of pure osteoblasts in a short period with no fibroblastic adulteration.