Quantitative study of tissue-engineered cartilage with human bone marrow mesenchymal stem cells

Objectives: To assess the possibility of cartilage tissue engineering using human mesenchymal stem cells (hMSCs) and to investigate the quantitative relationship between hMSCs and engineered cartilage. Design: Human mesenchymal stem cells were cultured, cryopreserved, and expanded in vitro. Surface antigens were detected by flow cytometry. In vitro chondrogenesis of hMSCs and cryopreserved hMSCs was performed. The chondrogenesis-induced hMSCs were seeded onto polyglycolic acid scaffolds, cultured in vitro for 3 weeks in chondrogenic medium, and then implanted into nude mice. The implants were harvested after 10 weeks and examined with histologic and immunochemical staining. Results: The construction of cartilages was identified grossly and histologically: 1.9 to 2.5 X 10(7) nucleated cells were obtained from 1 mL of bone marrow, and about 1 to 2 X 10(6) hMSCs were obtained from. the primary culture. The number of hMSCs tripled at every passage and reached 1.4 to 2.8 X 10(12) at passage 15. The purity of hMSCs was 95% and 98% at the primary and the fourth passages, respectively. Twenty-one days was the optimal (induction rate, 95%) induction time, with no apparent differences in induction rates among different passages. Based on our findings, hMSCs from 0.07 to 0.14 mL of bone marrow, expanded during 4 passages and induced for 21 days, would be sufficient to engineer 1 cm(2) of cartilage, 3-mm thick. Conclusion: Quantitative standards of hMSCs as seed cells for cartilage tissue engineering were established and may have value for later clinical work.