Method: Human bone marrow stem/stromal cells (hMSCs) were isolated per our prior published methods. Passage 2~3 hMSCs in monolayer or 3D pellet cultures were treated by sequential or combined connective tissue growth factor (CTGF) and transforming growth factor β3 (TGFβ3) for up to 4 wks. Fibrochondrogenic differentiation was evaluated using Alcian blue (AB) and Picrosirious red (PR) staining, and glycosaminoglycan (GAGs) and collagen (COL) were quantitatively assayed. Immunofluorescence was performed to identify cells expressing proCOL-I and/or proCOL-IIα. Single cell cloning was performed to appreciate the heterogeneity and specific lineage characteristics.
Result: Sequential or combined treatment of CTGF and TGFβ3 generated notable deposition of both GAG and collagen matrix. MSCs with sequential treatment of CTGF and TGFβ3 displayed significantly more pro-COL-I+/pro-COL-IIα+ cells than combinations of CTGF and TGFβ3. Certain clonal progenies (~55%) were capable of differentiating into fibrochondrogenic, osteogenic, chondrogenic, and adipogenic lineages. Single cell clones following sequential CTGF and TGFβ3 exposure simultaneously synthesized both type I and type II collagen.
Conclusion: These data provide the first original clue of derivation of fibrochondrocytes from bone marrow stem/stromal cells. Extending from our recent report of fibroblastic differentiation from MSCs by CTGF alone (Lee, et al., JCI 2010), the present findings suggest that sequential, but not concomitant, exposure of CTGF and TGFβ3 generates fibrochondrocytes. Together, these findings may have implications in a reproducible yield of fibrochonddrocytes for TMJ regeneration.
Keywords: Fibrochondrocytes, Joint dysfunction, Oral surgery, Regeneration and Tissue engineering
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