Hypoxia differentially regulates human nucleus pulposus and annulus fibrosus cell extracellular matrix production in 3D scaffolds G. Feng, L. Li, H.

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Hypoxia differentially regulates human nucleus pulposus and annulus fibrosus cell extracellular matrix production in 3D scaffolds G. Feng, L. Li, H. Liu, Y. Song, F. Huang, C. Tu, B. Shen, Q. Gong, T. Li, L. Liu, J. Zeng, Q. Kong, M. Yi, M. Gupte, P.X. Ma, F. Pei Osteoarthritis and Cartilage Volume 21, Issue 4, Pages 582-588 (April 2013) DOI: 10.1016/j.joca.2013.01.001 Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Morphology of IVD cells in two-dimensional (2D) culture. Passage 2 AF (A) and NP (B) cells on petri dishes. Osteoarthritis and Cartilage 2013 21, 582-588DOI: (10.1016/j.joca.2013.01.001) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Real-time PCR analysis of gene expression in NP and AF cells in 3D scaffolds. Constructs cultured for 1 week or 3 weeks under normoxic (20% O2) or hypoxic (2% O2) conditions. Statistical difference between results from hypoxic and normoxic cultures at each time point is shown. (Results show values for pooled samples, one pool per bar. *Denotes significance; exact P-values are reported in Results.) Osteoarthritis and Cartilage 2013 21, 582-588DOI: (10.1016/j.joca.2013.01.001) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Histological analyses of NP/AF cell-scaffold constructs cultured under hypoxia or normoxia for 3 weeks: (A–D) H&E staining; (E–L) Alcian blue staining. More abundant GAG amounts were detected in NP cell-scaffold constructs under hypoxic than under normoxic condition. No significant differences in the AF cell-scaffold constructs under different oxygen tension. Scale bar: 250 μm (A–H); 100 μm (I–L). Osteoarthritis and Cartilage 2013 21, 582-588DOI: (10.1016/j.joca.2013.01.001) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 IHC staining for collagen type I/II in AF/NP cell-scaffolds constructs at 3 weeks. Abundant collagen type II, while little collagen type I, was found in NP cell-scaffolds construct under hypoxia. In contrast, lower collagen type I indicated poor ECM formation in NP cell-scaffolds construct under normoxic condition. In AF cell-scaffolds constructs, abundant deposition of collagen type I was found either in normoxic or hypoxic condition. Osteoarthritis and Cartilage 2013 21, 582-588DOI: (10.1016/j.joca.2013.01.001) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 The GAG content was quantified for constructs cultured for 3 weeks. Hypoxia increased the accumulation of GAG in the constructs from NP cells, while AF cells were not affected. (Results show values for pooled samples, one pool per bar. *Denotes significance; exact P-values are reported in Results.) Osteoarthritis and Cartilage 2013 21, 582-588DOI: (10.1016/j.joca.2013.01.001) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 The DNA content was quantified for constructs cultured for 3 weeks. Hypoxia significantly increased DNA content of NP cells at 3 weeks. (Results show values for pooled samples, one pool per bar. *Denotes significance; exact P-values are reported in Results.) Osteoarthritis and Cartilage 2013 21, 582-588DOI: (10.1016/j.joca.2013.01.001) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Immunofluorescence staining for HIF-1α in cell-scaffold constructs after a 3-week culture: a significantly stronger fluorescent staining was found in NP cells cultured under the hypoxic condition (A) than the normoxic condition (B). Scale bar: 30 μm. Osteoarthritis and Cartilage 2013 21, 582-588DOI: (10.1016/j.joca.2013.01.001) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions