Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture Dr R.L. Mauck, Ph.D., X. Yuan, Dr.

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Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture Dr R.L. Mauck, Ph.D., X. Yuan, Dr R.S. Tuan, Ph.D. Osteoarthritis and Cartilage Volume 14, Issue 2, Pages 179-189 (February 2006) DOI: 10.1016/j.joca.2005.09.002 Copyright © 2005 Terms and Conditions

Fig. 1 Bovine MSCs undergo both adipogenesis and osteogenesis in confluent monolayer cultures after 21 days of exposure to adipogenic (B) or osteogenic (D) medium compared to those cultured in BM (A and C). (A and B) Oil Red O staining; (C and D) Alizarin Red staining. Scale bar: 50μm (A and B) or 200μm (C and D). Osteoarthritis and Cartilage 2006 14, 179-189DOI: (10.1016/j.joca.2005.09.002) Copyright © 2005 Terms and Conditions

Fig. 2 Analysis of cartilage phenotype in bovine chondrocyte and MSC cultures. (A) Chondrogenic gene expression in bovine chondrocytes (CH) or MSCs in monolayer (mono) culture and MSCs in pellet culture in CM with (+) or without (−) 10ng/mL TGF-β3. MSCs in pellet culture show a time-dependent increase in cartilage-specific matrix gene expression in the presence of TGF-β3. (B) Sections of Day 11 MSC pellets cultured in CM. a, b: Safranin O staining; c, d: Picrosirius Red staining. a, c: CM−; b, d: CM+; scale bar: 300μm. Osteoarthritis and Cartilage 2006 14, 179-189DOI: (10.1016/j.joca.2005.09.002) Copyright © 2005 Terms and Conditions

Fig. 3 Primary high-density culture of bovine chondrocytes (top left) and expansion of bovine bone marrow derived MSCs (top right) demonstrating differences in cellular morphology. Scale bar: 100μm. Bovine chondrocytes and MSCs in 3D agarose culture take on a rounded morphology and remain viable after encapsulation. Bright-field (middle) and LIVE/DEAD (bottom) images of bovine chondrocytes (left) and MSCs (right) 24h after encapsulation at a concentration of 20 million cells/mL. Scale bar: 50μm (middle) or 100μm (bottom). Osteoarthritis and Cartilage 2006 14, 179-189DOI: (10.1016/j.joca.2005.09.002) Copyright © 2005 Terms and Conditions

Fig. 4 Alcian Blue staining of Day 70 constructs reveals the formation of a proteoglycan-rich cartilaginous matrix in both chondrocyte- (first column) and MSC- (third column) laden constructs with differing medium compositions. Scale bar: 100μm. Cell viability of constructs analyzed by LIVE/DEAD staining of chondrocyte- (second column) and MSC- (fourth column) laden constructs after 11 weeks of culture with differing medium compositions. Chondrocytes are viable in every culture condition, with evidence of cell division in CM+. Bovine MSCs remain viable in 3D culture only when cultured in CM. Scale bar: 100μm. Osteoarthritis and Cartilage 2006 14, 179-189DOI: (10.1016/j.joca.2005.09.002) Copyright © 2005 Terms and Conditions

Fig. 5 sGAG content (%ww), bulk collagen content (%ww), and DNA content (DNA∗1000 (per ww)) of chondrocyte- and MSC-laden constructs in free swelling culture in different media over a 10-week time course. In CM+, chondrocyte-laden constructs show larger increases in sGAG and DNA content than MSC-laden constructs, while similar bulk collagen levels are achieved in each cell type. CM+: CM with TGF-β3; CM−: CM without TGF-β3. Data represent the mean and standard deviation of three to nine samples per group. *Indicates P<0.05 vs Day 0 within group; **indicates P<0.05 between groups on Day 70. Osteoarthritis and Cartilage 2006 14, 179-189DOI: (10.1016/j.joca.2005.09.002) Copyright © 2005 Terms and Conditions

Fig. 6 Equilibrium compressive Young's modulus (EY−) and dynamic modulus (ED, 1Hz) of chondrocyte- and MSC-laden constructs in free swelling culture in different media over a 10-week time course. Increases in mechanical properties are most prominent in cultures maintained in CM+, with chondrocyte-laden constructs achieving a higher stiffness than MSC-laden constructs similarly maintained. CM+: CM with TGF-β3; CM−: CM without TGF-β3. Data represent the mean and standard deviation of three to nine samples per group. *Indicates P<0.05 vs Day 0 within group; **indicates P<0.05 between groups on Day 70. Osteoarthritis and Cartilage 2006 14, 179-189DOI: (10.1016/j.joca.2005.09.002) Copyright © 2005 Terms and Conditions