Chondrogenic differentiation of human bone mesenchymal stem cells treated with growth differentiation factor 5 under hypoxia

Abstract

ObjectiveTo explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMSCs) induced by growth differentiation factor-5 (GDF-5) under hypoxia.Methods After hBMSCs were isolated from ilium, flow cytometry was performed to detect the cell surface markers, hBMSCs were exposed to cartilage inducing medium containing 100 ng/mL of GDF-5 in hypoxic (2％ oxygen) and normoxic (21％ oxygen)conditions, respectively. RT-PCR was performed to detect the expressions of type Ⅱ collagen and aggrecan in the cells after 3 weeks. In hypoxic and normoxic conditions, the hBMSCs were induced in conditioned medium (CM) containing GDF-5 for 3 weeks. After digestion, the hBMSCs, according to a certain density, were inoculated on 24-well plates coated with 2％ of agarose. Induction in the CM continued for another 3 weeks.Examples of both groups were detected for types Ⅰ and l collagen with histology and immunohistochemistry.Toluidine blue staining was used to detect the expressions of cartilage-specific components.Results The hBMSCs grew in a manner of spindle-shaped whirlpool, with high expressions of CD44 and CD29 and a negative expression of CD45. The hBMSCs exposed to the CM containing GDF-5. in the hypoxic condition, expressed type Ⅱ collagen and aggrecan significantly more than those exposed to the chondrogenic growth factors in the nonnoxic condition ( P ＜ 0. 05). The hBMSCs, in the hypoxic condition, after induction by GDF-5,inoculation on agar surface and induction in the CM, were self-assembled to form cartilage-like tissue in proper shape and size. In the hypoxia group, the expression of type Ⅱ collagen was increased and the expression of type Ⅰ collagen was decreased. The toluidine blue staining in the hypoxia group was deeper than in the normoxia group.ConclusionsHypoxia may promote differentiation of hBMSCs into chondrocytes.The ” self-assembly” technology can make it possible that chondrogenically differentiated hBMSCs induced by GDF-5 under hypoxia can be built in vitro into successful tissue-engineered cartilage. Key words: Hypoxia, cellular; Chondrocytes; Bone marrow cells