Background Injectable three-dimensional （3D） scaffolds have the advantages of fluidity and moldability to fill irregularshaped defects,simple incorporation of bioactive factors,and limited surgical invasiveness.Adipose-derived stem cells （ADSCs） are multipotent and can be differentiated toward nucleus pulposus （NP）-Iike cells.A hypoxic environment may be important for differentiation to NP-like cells because the intervertebral disc is an avascular tissue.Hence,we investigated the induction effects of hypoxia and an injectable 3D chitosan-alginate （C/A） gel scaffold on ADSCs.Methods The C/A gel scaffold consisted of medical-grade chitosan and alginate.Gel porosity was calculated by liquid displacement method.Pore microstructure was analyzed by light and scanning electron microscopy.ADSCs were isolated and cultured by conventional methods.Passage 2 BrdU-labeled ADSCs were co-cultured with the C/A gel.ADSCs were divided into three groups （control,normoxia-induced,and hypoxia-induced groups）.In the control group,cells were cultured in 10％ FBS/DMEM.Hypoxia-induced and normoxia-induced groups were induced by adding transforming growth factor-β1,dexamethasone,vitamin C,sodium pyruvate,proline,bone morphogenetic protein-7,and 1％ ITS-plus to the culture medium and maintaining in 2％ and 20％ O2,respectively.Histological and morphological changes were observed by light and electron microscopy.ADSCs were characterized by flow cytometry.Cell viability was investigated by BrdU incorporation.Proteoglycan and type Ⅱ collagen were measured by safranin O staining and the Sicool method,respectively.mRNA expression of hypoxia-inducing factor-1α （HIF-1α）,aggrecan,and Type Ⅱ collagen was determined by reverse transcription-polymerase chain reaction.Results C/A gels had porous exterior surfaces with 80.57％ porosity and 50-200 μm pore size.Flow cytometric analysis of passage 2 rabbit ADSCs showed high CD90 expression,while CD45 expression was very low.The morphology of induced ADSCs resembled tha
Zhang Zhicheng Li Fang Tian Haiquan Guan Kai Zhao Guangmin Shan Jianlin Ren Dajiang
