Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors.

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Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion W.L. Grayson, S. Bhumiratana, P.H. Grace Chao, C.T. Hung, G. Vunjak-Novakovic Osteoarthritis and Cartilage Volume 18, Issue 5, Pages 714-723 (May 2010) DOI: 10.1016/j.joca.2010.01.008 Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Experimental design. (A) Biphasic scaffold made by interfacing agarose and trabecular bone scaffolds. (B) Perfusion bioreactor for cultivation of biphasic scaffolds. Enlarged view shows predicted path of medium flow through the scaffolds and through the sides into the reservoir. (C) Schematic of experimental design. Osteoarthritis and Cartilage 2010 18, 714-723DOI: (10.1016/j.joca.2010.01.008) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Cell differentiation studies. (A–C) Morphology of hMSCs cultured using (A) expansion medium, (B) osteogenic supplements and (C) chondrogenic supplements. (D–F) Von Kossa staining of pellets cultured under osteogenic conditions for 4 weeks. (G–I) Alcian Blue staining of pellets cultured under chondrogenic conditions for 4 weeks. Osteoarthritis and Cartilage 2010 18, 714-723DOI: (10.1016/j.joca.2010.01.008) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Quantitative properties of cartilage region. (A) GAG content of gels normalized by wet weight. (B) Equilibrium modulus. (C) DNA content normalized by wet weight. (D) BV of gels measured by μ-CT. (n=3; *P<0.05; **P<0.001) (E) Table of GAG expression and equilibrium modulus values normalized to DNA (n=3; *P<0.05 as compared to the experimental groups using the same culture medium). Osteoarthritis and Cartilage 2010 18, 714-723DOI: (10.1016/j.joca.2010.01.008) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Representative images of immunohistochemical properties of cartilage region. First row: H&E staining. Second row: Alcian Blue staining for GAG content. Third row: collagen II expression. Fourth row: collagen×expression. Fifth row: BSP expression. Osteoarthritis and Cartilage 2010 18, 714-723DOI: (10.1016/j.joca.2010.01.008) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Representative images of immunohistochemical properties of bone region. First row: H&E staining. Second row: Alcian Blue staining for GAG content. Third row: collagen I expression in gels. Fourth row: BSP expression. Osteoarthritis and Cartilage 2010 18, 714-723DOI: (10.1016/j.joca.2010.01.008) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Integration of bone and cartilage regions. (A) Integration region of static constructs (UD-S-C) is mostly acellular. (B) High magnification image of region indicated by box in (A) showing different morphologies in gel and scaffold regions and minimal GAG expression. (C) Integration between cartilage and bone is enhanced under bioreactor conditions. (D) High magnification image of region indicated by box in (C) shows high matrix production in central regions of integration zone. Distinct spherical morphology in gel is indicated by * whereas the elongated, fibrous morphology is evident in regions close to bone by arrows (↑). Osteoarthritis and Cartilage 2010 18, 714-723DOI: (10.1016/j.joca.2010.01.008) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions