Objective      To investigate a new method of isolating human umbilical cord mesenchymal stem cells (hUC-MSCs) by explants culture and explore their features as seed cells in tissue engineering bone (TEB).       Methods      Fresh sterile umbilical cord tissues were obtained afte

r approval of ethics committee of our hospital and signature of informed consent. The tissues containing more impurities cells were removed to make sure that the remained tissue as much as possible for Wharton’s jelly. Tissue culture dish (experiment group) and common culture dish (control group) were used to isolate UC-MSCs from the umbilical cord tissues. The successful rate of tissue culture and the number of primary cells were noted. Flow cytometry was used to detect the expression of cell surface molecules

and the potentials of multi-directional differentiation in osteogenicity

chondrogenicity

and adipogenicty in P3 cells. The obtained P3 cells were used to construct the TEB with demineralized bone matrix (DBM). The cell adhesion was detected. The growth of the cells was observed by laser confocal microscopy

and the amount of total proteins were also determined.       Results      In 12 d after culture

significant differences were seen between the experiment group and control in the number of floating explants (0 vs 13

P0.01)

successful culture rate[(86.0±2.7)% vs (44±4)%

P0.01

]

and number of cells[(24.7±3.6)×10

4

vs (5.1±1.0)×10

4

P0.01

]

. The obtained P3 cells were positive to CD44

CD73

CD90 and CD105

+

and negative to CD34 and CD45

with osteogenic

chondrogenic

and adipogenic differentiation capabilities. The cells had an adhesive rate of (81.3±4.7)%

exhibited good compatibility

and grew well and proliferated with time on TEB.       Conclusion       Tissue culture dish is a simple and standardized procedure to isolate hUC-MSCs

with high utilization rate

and harvests abundant cells (increased by about 15 to 20 times). Those cells have very good compatibility with DBM

and TEB are successfully constructed in vitro.