Comparative Study On Osteogenic And Chondrogenic Potential Between Dedifferentiated Adipocytes And Adipose Derived Stem Cells

Objective:To compare the chondrogenic and osteogenic potential of rabbit dedifferentiated adipocytes (DA) and adipose derived stem cells (ADSCs) in vitro, and to provide the basis for the choice of seed cells for the Bone tissue engineering.Methods:①ADSCs and Mature adipocytes (MA) were isolated from the subcutaneous adipose tissue which was from4-month-old New Zealand white rabbits’abdomen by mechanical digestion and enzyme digestion, and then cultured and amplified in vitro.②Inverted ceiling adherent culture method was used to make the adipocytes dedifferentiate and to obtain the DA.③ADSCs and DA were seed into24-well plates (2x104cells/well) separately. The morphology and proliferation of both ADSCs and DA were observed using inverted phase contrast microscope in the whole time. The numbers of both kind of cell proliferation were detected using cell count assay every day, and the mean numbers of which were used to draw the growth curve from primary to the passage3and obsere the doubling time.④The passage3of ADSCs and DA were used for the induction of chondrogenesis and osteogenesis. The experiment was divided into6groups:ADSCs cultured under condition of osteogenic media (Ao); DA cultured under condition of osteogenic media (Do); ADSCs cultured under condition of chondrogenic media (Ac); DA cultured under condition of chondrogenic media (Dc); Each group had its control group. After cultured for14days, alizarin red and type I collagen immunohistochemical staining were used to detect osteoblasts in Ao and Do group while the chondrocytes of Ac and Dc group were detected by collagen type II immunohistochemistry staining and proteoglycan toluidine blue staining.⑤The cells cultured14days and21days were used to detect the expression of collagen-I mRNA by semi-quantitative RT-PCR in group Ao and Do,and the obtained data was compared using t test line between and within group, while the same way was used to detect collagen-II mRNA expression and obtain the statistical analysis in Ac and Dc group. The chondrogenic and osteogenic potential of ADSCs and DA were compared.Results:①Primary MA showed a regular ring-like small circular morphology, and was aggregated distribution. By the inverted ceiling adherent culture method, the lipids of MA removed from the cytoplasm gradually, and the morphology of the cells changed from circle into ellipse, and performed as long shuttle fibroblast-like formation. Primary ADSCs were long spindle-shaped or small polygon, uniform in size and diffuse distribution.②After subculture, both two kind of cells were long spindle-shaped morphology, densely distributed, and "whirlpool-like" growth.③The terminal cell numbers of primary passage in ADSCs at logarithmic growth stage was5.9±0.41, while the DA was6.3±0.36, and there were no significant statistical differences between the ADSCs and DA primary passage cells (p>0.05).The same results were obtained at the2nd and3rd passage of this two kind of cell (p>0.05).The doubling time of both ADSCs and DA were among58to61h (p>0.05),showing a similar proliferative ability.⊙Alizarin red and type I collagen immunohistochemical staining in Ao and Do group were positive, while in the contol groups were negative. The collagen type Ⅱ immunohistochemistry staining and proteoglycan toluidine blue staining of Ac and Dc groups showed positive results, and the results of their control groups were negative.⑤The expressions of collagen-I mRNA detected by semi-quantitative RT-PCR in group Ao at the14th and21th day were0.375±0.018and0.908±0.017,while in group Do were0.246±0.014、0.821±0.012. The expressions of collagen-I mRNA in group Ao at both14th and21th day were significantly higher than group Do (p<0.05) and also the expression of type I collagen mRNA was proportional to the incubation time in both group Ao and Do (p<0.05). The expressions of collagen-Ⅱ mRNA in group Ac and Dc at the14th and21th day were0.316±0.027、0.458±0.020and0.204±0.024、0.377±0.021. The expressions of collagen-Ⅱ mRNA in group Ac at the14th and21th day were higher than group Dc (p<0.05),and there was a positive correlation between the expression of collagen-Ⅱ mRNA and the incubation time in this two group (p<0.05). The expression of collagen-Ⅰ and collagen-Ⅱ mRNA in all the control groups were negative, which were significant difference compared with the experiment (p<0.05) Conclusion:MA could be induced to dedifferentiate into DA by the inverted ceiling adherent culture method. The morphology of DA and ADSCs had no obvious variation in vitro. There were similar proliferative ability between the DA and ADSCs. Both of them could be induced to osteogenic and chondrogenic differentiation, but ADSCs seemed to be more potential. Both of the DA and ADSCs could be considered as seed cells in the Bone tissue engineering.