[Influence of co-culturing vascular endothelial cells and adipose-derived stromal cells on osteogenic differentiation in vitro].
OBJECTIVE
To further study the influence of the co-cultivation of vascular endothelial cells (VECs) and adipose-derived stromal cells (ADSCs) on cell osteogenic differentiation in vitro and provide experimental evidences of the probability of the co-cultivation of VECs and ADSCs as the seed cells of tissue engineering.


METHODS
The VECs derived from cord blood and ADSCs were prepared by full-term pregnancy SD rats and 18-week-old SD rats, to carry on the morphological observation and immunohistochemical staining identification. The third generation of ADSCs and the VECs induced by conditioned medium for 6 weeks were cultured and were divided into groups A, B, and C as the experimental group according to cell ratios of 3 : 1, 1 : 1, and 1 : 3, respectively. ADSCs or VECs was cultured alone in groups D and E as control groups. ALP and alizarin red staining were done respectively on the 7th day and 14th day; ALP and osteocalcin (OC) were detected respectively on the 4th day, 7th day, and 14th day.


RESULTS
The VECs derived from cord blood showed mixed growth of short spindle and polygonal cells after 6 weeks of induction, the immunofluorescent staining result of von Willebrand factor was positive. ADSCs showed adherent mononuclear cells and spindle-shaped growth without duplication; the immunofluorescent staining result of CD90 was positive and no positive cells were seen in the control group. On the 7th day of cell culture, ALP staining showed that the results were negative in groups A, D, and E, and some positive cells were seen in groups B and C; on the 14th day, the results were still negative in groups D and E, and positive cells fused to sheet form in groups A, B, and C. von Kossa staining showed that the results were negative in all groups on the 7th day; few positive cells were seen in groups A, B, and C, and no positive cells were seen in groups D and E on the 14th day. The ALP contents increased gradually in all groups, which was highest in group B at every time point, showing significant difference (P < 0.01) between group B and other groups, between groups A, C and groups D, E. The OC value increased gradually in every group, which was highest in group B on the 7th and 14th days, showing significant difference between group B and other groups (P < 0.01), between group C and group D (P < 0.01) on the 4th and the 14th days, between groups A, C and group E (P < 0.05) on the 14th day.


CONCLUSIONS
ADSCs have potential of osteogenic differentiation by VECs in the system of co-culturing VECs and ADSCs in vitro, the influence on osteogenic differentiation is the strongest in a ratio of 1 : 1.
Wang
Fuke
F
Department of Head and Neck Surgery, the 1st Affiliated Hospital of Kunming Medical College, Kunming Yunnan, 650032, P.R. China.
Liu
Liu
L
Zhao
Deping
D
He
Xiaoguang
X
Dai
Xiaoming
X
Li
Yisong
Y
Li
Yuxiao
Y
D017667:Adipocytes / Q000166:cytology*; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology*; D005260:Female; D010012:Osteogenesis; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D017154:Stromal Cells / Q000166:cytology*