Bone Regeneration Using Autologous Adipose-Derived Stem Cell Spheroid Complex

Abstract

Bone defects require reconstruction using various biomaterials or non-biological materials. Stem cell spheroids can be used for scaffold-free approaches for osteogenesis. We set up a culture method for creating an optimal osteogenic adipose-derived stem cell (ADSC) spheroid complex by measuring the expression of protein in a sequential series of culture media. After culturing ADSC spheroids for 24 hours in DMEM, the spheroids were cultured in ascorbic acid-containing medium for five days followed by osteoblast differentiation medium. One day after exchanging to osteoblast differentiation medium, spheroids were collected and cultured for four days to obtain a spheroid complex. Each culture period was determined by analyzing the expression of collagen type I, alkaline phosphatase and integrin α5 to maximize the activity of ADSC spheroids. The expression of collagen type I increased significantly in ascorbic acid-containing medium (p < 0.05) compared with control medium on day five, suggesting that culturing spheroids in ascorbic acid increases collagen synthesis. RNA was extracted from ADSC spheroids after 1, 3, 5, and 7 days in each medium and RT-PCR was performed to measure integrin α5 expression. The expression was transiently high on the first day of osteoblast differentiation culture and then gradually decreased. Osteoblast differentiation medium enhanced cell adhesion in spheroids. An in vivo study confirmed the osteogenic potential of the ADSC spheroid complex created by the established protocol. The ADSC spheroid complex stimulated bone regeneration and will be applied to the treatment of large bone defects.