BACKGROUND: The nucleus pulposus tissue engineering is regarded as an ideal method in repairing intervertebral disc degeneration. However, the resource of nucleus pulposus cells is limited, so seed cell plays a vital role in nucleus pulposus (NP) tissue engineering. OBJECTIVE: To verify whether adipose-derived stem cells (ADSCs) can differentiate into NP-like cells induced by three-dimensional scaffold. DESIGN, TIME AND SETTING: An in vitro observation based on cell-materials level was performed at the Orthopaedic Research Institute, General Hospital of Beijing Military Area Command of Chinese PLA from July to December 2008. MATERIALS: Three Japanese big-eared rabbits with 3-months old were provided by Vital River Laboratory Animal Technology Co., Ltd., and the medical chitosan was purchased from Golden-shell Biochemical Co., Ltd. METHODS: The ADSCs were isolated from the rabbit adipose and then subcultured with collagenase digestion. The second ADSCs were prepared 50 uL cell suspension (5×10~6 cells), and mixed with 150 uL chitosan-alginate (C/A) and cultured in a12-well plate for 20 minutes in room temperature to obtain C/A gel. The experiment divided into 3 groups, in the control group, 2 mL DMEM containing 10% fetal bovine serum was added; DMEM containing bovine serum albumin, transforming growth factor-1 (TGF), hexadecadrol, vitamin C, bone morphogenetic protein-7,1% ITS was added, and then cultured with 2% O_2 or 20% O_2. All the samples were placed in incubator at a humidified atmosphere of 5% O_2 at 37 V for 21 days. MAIN OUTCOME MEASURES: Histomorphological observation; levels of proteoglycan, as well as type II collagen. RESULTS: The scanning electron microscopy showed that ADSCs grew well on the chitosan-alginate scaffolds, which was positive to oil-O staining and Alcian blue staining. More proteoglycan was produced. With time increasing, there was no significant change in the proteoglycan content and type II collagen in the control group. The content of proteoglycan and collagen type II was higher in the induced group, which had difference to the control group (P < 0.001, P < 0.05). Meantime, the expression of collagen type II and proteoglycan was greater in hypoxia-induced group than that of normoxia-induced group (P<0.05, P<0.01). CONCLUSION: Rabbit ADSCs growing on the scaffolds of chitosan-alginate gel may differentiate towards NP-like cells under certain conditions, especially better under hypoxia state.

sed by pelvic fracture or other conditions.