Experimental Study On Osteogenic Differentiation Of Adipose-derived Adult Stem Cells Labeled By BrdU

Bone defect and nonunions caused by trauma,tumor resection and infection are often been seen in orthopedics clinic, and it is difficult to cure them. In recent years, with the rapid development of tissue engineering technology, attempting to cure bone defect and nonunions by bone tissue engineering has become the centre of research in the field. Bone tissue engineering mainly includes seed cell,scaffold and growth factor. Among these elements, seed cell is essential for bone tissue engineering, and is also the prerequisite for further development of bone tissue engineering. Obtaining one ideal seed cell which owns sufficient quantity and favorable function has been an urgent task for bone tissue engineering. In our study, we isolated a new stem cells-adipose-derived adult stem cells(ADASCs). We labelled ADASCs with BrdU, then induced them osteogenesis in vitro and planted them in vivo, and then observed their differentiation, made sure whether it was feasible to apply ADASCs in bone tissue engineering extensively.Objective:At first we studied the feasibility of labeling ADASCs which were isolated from rabbit's adipose tissue and cultured in vitro with BrdU. If BrdU was able to label ADASCs, we induced ADASCs which were labeled by BrdU osteogenesis in vitro, then planted them in vivo and traced them. Thus we could investigate the feasibility of ADASCs being applied in bone tissue engineering extensively as seed cell.Methods:1.Labeling ADASCs with BrdU in vitroADASCs were isolated from rabbit's subcutaneous adipose tissue which was minced and digested by collagenase Type I.Then they were cultured and expanded in control medium. The third passage was incubated by BrdU at 25μmol/L for 48h when cells confluenced about 50%. Then immunohistochemical assay was performed to observe the labeling of ADASCs. Then by the morphology under the microscope,trypanblau testing,MTT testing, we surveyed the influence of BrdU on the growth of ADASCs.2. Tracing osteogenic differentiation of ADASCs in vivo with BrdUBased on the assurance that BrdU was able to label ADASCs,we labeled ADASCs with BrdU, then cultured them to the third passage. We incubated the third passage cells in induced medium in order to induce them osteogenesis.Then we examined the effect by alkaline phosphatase,alizarin red stain and collagen I immunocytochemistry. After 2w, ADASCs were seeded onto rabbit cancellous bone(RCB). The ADASCs/RCB complexs were cultured in induced medium for 7 days in vitro, and the growth of ADASCs on RCB was observed by scanning electron microscope(SEM). Then the complexs were planted into the muscles of rabbit which produced ADASCs. After 4w,8w and 12w, we killed rabbits and took out of ADASCs/RCB complexs. HE stain was performed to observe the formation of new bone. BrdU immunocytochemistry was performed to detect the osteogenic differentiation of ADASCs in vivo. Results:1. BrdU immunocytochemistry after ADASCs being incubated by BrdU showed that BrdU was able to label ADASCs successfully, and the labeling rate reached (82.3±8.6)%. The growing shapes of cells under the microscope between ADASCs which were labeled and not labeled were similar. Trypanblau testing showed that the survival rate of ADASCs which were labeled by BrdU was (94.4±1.5)%, and the survival rate of ADASCs which were not labeled was (95.2±1.2)%. There was no significant difference between them(P>0.05). So BrdU made no influence on the activity of ADASCs. MTT testing showed that there was no significant difference at each time spot between ADASCs which were labeled and not labeled (P>0.05). The proliferation tendencies of cells between the two groups were also similar.2. The expressions of Alkaline phosphatase,alizarin red stain and collagen I immunocytochemistry after ADASCs being cultured in induced medium in vitro were all positive, which showed that ADASCs were induced osteogenesis successfully. SEM examination after ADASCs being combined with RCB showed that ADASCs grew well on RCB. HE staining after ADASCs/RCB complexs being taken out from rabbits showed that there was no new bone formation at 4w, while there was definite new bone formation at 8w and 12w. BrdU immunocytochemistry showed that there were positive ADASCs in new bone, which illustrated that ADASCs labeled by BrdU could form new bone in vivo.Conclusions:BrdU is feasible and ideal to label ADASCs. Thus we can make use of BrdU to label ADASCs and trace their differentiation in vivo.After being induced osteogenesis in vitro and planted in vivo, ADASCs can keep osteogenic differentiation in vivo and form new bone furtherly. Thus we confirm the osteogenic capability of ADASCs in vivo and provide the important evidence of applying ADASCs in bone tissue engineering extensively as seed cell.