张 波(Zhang Bo，Dept Orthop，Union Hosp，Tongji Med Col，Huazhong Sci Technol Univ，Wuhan 430022)…∥Chin J Orthop Trauma.－2011，13(2).－160～164

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

张 波(Zhang Bo，Dept Orthop，Union Hosp，Tongji Med Col，Huazhong Sci Technol Univ，Wuhan 430022)…∥Chin J Orthop Trauma.－2011，13(2).－160～164

ObjectiveTo explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells(hBMCs)induced by growth differentiation factor-5(GDF-5)under hypoxia.MethodsAfter 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 expression of typeⅡcollagen and aggrecan in the cells after 3 weeks.In hypoxic and normoxic conditions，the hBMSCs were induced in conditioned medium(CM)containingGDF-5for 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 typesⅠandⅡcollagen with histology and immunohistochemistry.Toluidine blue staining was used to detect the expressions of cartilage-specific components.ResultsThe 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 be CM containing GDF-5，in the hypoxic condition，expressed typeⅡcollagen and aggrecan significantly more than those exposed to the chondrogenic growth factors in the normoxic 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 typeⅠcollagen was decreased.The toluidine blue staining in the hypoxia group was deeper than in the normoxia group.ConclusionHypoxia 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 builtin vitrointo successful tissue-engineered cartilage.12 refs，4 figs，1 tabs

(Authors)