Establishment And Evaluation Of Culture Methods For Osteoblasts Derived From Female Suckling Rat Skulls
Keywords
? ,Collagenase,Trypsin,Osteoblast,Cell Culture,? , Collagenase, Trypsin, Osteoblasts, Cell Culture
Abstract
Objective: To explore the best method for establishing in vitro culture of neonatal mouse osteoblasts and to provide a stable and reliable source of seed cells for bone tissue engineering research. Methods: The skulls of neonatal mice (within 48 hours) were used to culture cells in vitro using a modified two-step digestion method of trypsin and collagenase. The cell morphology was observed under an inverted microscope, and the alkaline phosphatase (ALP) activity and mineralization ability were observed. Identify and draw cell growth curves to observe the phenotypic characteristics of osteoblasts. Results: Primary cells inoculated 24? After 1 hour, some round cells can be seen adhering to the wall in a scattered manner. At 48 to 72 hours, they completely adhere to the wall and stretch into a plump and polygonal shape, forming multiple colonies. At 7 to 8 days, the number of colonies increases rapidly. By about 10 to 15 days, the cells enter the rapid growth phase. The cells in the center of the colony are densely packed, and the surrounding cells are arranged in a radial or swirling pattern. There is no contact inhibition between cells, and there is overlap between colonies; the fused cells grow in overlapping and stratified layers after 15 to 20 days. The morphology of cultured osteoblasts shows irregular polygons, mostly irregular triangles or quadrilaterals, with short and thin protrusions on the surface. Hematoxylin staining, alkaline phosphatase staining and alizarin red staining of mineralized nodules confirmed that they had a complete osteoblastic phenotype and were indeed osteoblasts. Conclusion: The improved collagenase and trypsin combined digestion method can obtain high-purity osteoblasts in a short time, and is expected to become a routine method for seed cell culture in bone tissue engineering. ª£
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Original research was done by Na Jian, Ma Chao, Huo Weiling, Qin Ruiyun, Xu Yong, Wang Tao, Wu Xiaodong, Gu Guishan
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