Sox9Potentiates Bmp2-induced Chodrogenic Differentiation And Inhibits Bmp2-induced Osteogenic Differentiation

Background/Aims: There is a great clinical need to develop tissueengineering cartilage for effective cartilage regeneration. Bonemorphogenetic protein2(BMP2) has been confirmed as one of the mostchondrogenic growth factors, but it also exhibits osteogenic abilities andtriggers endochondral ossification. Effective chondrogenesis and inhibitionof BMP2-induced osteogenesis and endochondral ossification can beachieved by directing the mesenchymal stem cells (MSCs) towardschondrocyte lineage with chodrogenic factors, such as Sox9. Here weinvestigated the effects of Sox9on BMP2-induced chondrogenic andosteogenic differentiation of MSCs.Methods：(1) BMP2and Sox9gene expression were mediated by recombinantadenoviruses AdBMP2and AdSox9, AdGFP was used as a vector control.Then the adenoviruses were amplified in HEK293cells. C3H10T1/2cellswere infected by AdGFP, AdBMP2or AdSox9and the gene expression of BMP2and Sox9were detected by semi-quantitative RT-PCR. Western-blotanalysis was used to determine the expression of Sox9in each treatmentgroups at desired time points.(2) C3H10T1/2cells transduced with AdGFP, AdBMP2and/orAdSox9were cultured in micromass culture for2to14days in vitro. Alcianblue staining and quantification were applied to detect sulfatedglycosaminoglycan in cellular matrix at desired time points. QPCR was usedto determine the gene expression of ACAN and Col2a1at different timepoints. Col2a1protein expression was determined by Western-blot analysisat at continuously time points.(3) C3H10T1/2cells transduced with AdGFP, AdBMP2and/orAdSox9were cultured in monolayer culture in vitro. ALP activities weremeasured using ALP histochemical staining and quantification.Immunocytochemical staining was applied to detect OPN protein expressionin cellular matrix. QPCR was used to determine the gene expression ofRunx2, OPN and OC at different time points. OPN and OC proteinexpression were determined by Western-blot analysis at desired time points.(4) Ectopic cartilage/bone formation assay was used to further evaluatethe effect of Sox9on BMP2induced C3H10T1/2cells chondrogenic andosteogenic differentiation.(5) Mouse fetal limb explant culture was applied to evaluate the effectof Sox9on BMP2induced chondrocyte proliferation, hypertrophy and ossification.Results:(1) C3H10T1/2cells could be infected by adenoviruses and expressthe target gene effectively. AdBMP2could induce Sox9expression，whencombined with AdSox9, it showed a significant earlier onset ofoverexpression of Sox9and maintained significant values at high levelcontinuously.(2) Exogenous overexpression of Sox9enhanced BMP2-inducedchondrogenic differentiation of MSCs in a sequential time points inmicromass culture in vitro.(3) Exogenous overexpression of Sox9inhibited BMP2induced earlyand late osteogenic differentiation of MSCs in vitro.(4) Ectopic cartilage/bone formation assay demonstrated Sox9potentiated BMP2-induced cartilage formation and inhibited BMP2-inducedendochondral ossification.(5) Mouse limb cultures indicated that BMP2and Sox9actedsynergistically to stimulate chondrocytes proliferation and Sox9inhibitedBMP2-induced chondrocytes hypertrophy and ossification.Conclusion: This study strongly suggests that Sox9potentiatesBMP2-induced MSCs chondrogenic differentiation and cartilage formation,and inhibits BMP2-induced MSCs osteogenic differentiation andendochondral ossification. Thus, exogenous overexpression of Sox9in BMP2-induced mesenchymal stem cells differentiation may be a newstrategy for cartilage tissue engineering.