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. 2011;6(8):e23119.
doi: 10.1371/journal.pone.0023119. Epub 2011 Aug 15.

Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors

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Free PMC article

Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors

Kifah Shahin et al. PLoS One. .
Free PMC article

Abstract

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min(-1)) and gradually increasing (0.075-0.2 mL min(-1)) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0-4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min(-1). GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Properties of cartilage constructs produced using PGA scaffolds cultured in shaking T-flasks (non-perfused control), in perfusion bioreactors using a constant flow rate of 0.2 mL min−1 (high flow rate), a constant flow rate of 0.075 mL min−1 (low flow rate), or a gradually increasing flow rate of 0.075–0.2 mL min−1 (gradual increase in flow rate), or using scaffold pre-culture in T-flasks for either 5 days (5-day pre-culture) or 2.5 weeks (2.5-week pre-culture) prior to bioreactor culture at a constant flow rate of 0.2 mL min−1.
(a) Construct wet weight; and (b) number of cells. The scaffolds were seeded using 20×106 cells and cultured for a total of 5 weeks after seeding. The error bars represent standard errors from triplicate T-flask and/or bioreactor cultures. For each construct property, results labeled with different letters (A, B, C) are statistically different from each other (p<0.01).
Figure 2
Figure 2. Biochemical properties of cartilage constructs produced using PGA scaffolds cultured in shaking T-flasks (non-perfused control), in perfusion bioreactors using a constant flow rate of 0.2 mL min−1 (high flow rate), a constant flow rate of 0.075 mL min−1 (low flow rate), or a gradually increasing flow rate of 0.075–0.2 mL min−1 (gradual increase in flow rate), or using scaffold pre-culture in T-flasks for either 5 days (5-day pre-culture) or 2.5 weeks (2.5-week pre-culture) prior to bioreactor culture at a constant flow rate of 0.2 mL min−1.
(a) GAG concentration; (b) total collagen concentration; (c) collagen type II concentration; and (d) collagen type II as a percentage of total collagen. The scaffolds were seeded using 20×106 cells and cultured for a total of 5 weeks after seeding. The error bars represent standard errors from triplicate T-flask and/or bioreactor cultures. n.a. = not analyzed. For each construct property, results labeled with different letters (A, B, C, D) are statistically different from each other (p<0.01).
Figure 3
Figure 3. Histological appearance of constructs produced using PGA scaffolds cultured in bioreactors for 5 weeks at a constant flow rate of 0.2 mL min−1 (high flow rate: a, c, e, g) or gradually increasing flow rate of 0.075–0.2 mL min−1 (gradual increase in flow rate: b, d, f, h).
The scaffolds were seeded using 20×106 cells. Construct cross-sections show: (a, b) pink–red staining for GAG; (c, d) immunostaining (brown) for collagen type I; (e, f) immunostaining (brown) for collagen type II; and (g, h) immunostaining (brown) for collagen type II and blue–purple staining for cells at high magnification.
Figure 4
Figure 4. GAG release into the medium and retention in the constructs for PGA scaffolds cultured in bioreactors operated using a constant flow rate of 0.2 mL min−1 (high flow rate, •), a constant flow rate of 0.075 mL min−1 (low flow rate, ○), a gradually increasing flow rate of 0.075–0.2 mL min−1 (gradual increase in flow rate, □), or scaffold pre-culture in T-flasks for either 5 days (5-day pre-culture, ▪) or 2.5 weeks (2.5-week pre-culture, ▵) prior to bioreactor culture at a constant flow rate of 0.2 mL min−1.
(a) Cumulative amount of GAG released into the medium; (b) overall specific rate of GAG release (mg per day per mg of GAG in the constructs at harvest); and (c) percentage of total GAG (construct+medium) retained in the constructs. The scaffolds were seeded using 20×106 cells and cultured for a total of 5 weeks after seeding. The error bars represent standard errors from triplicate bioreactor cultures. Medium GAG data for the 5-day pre-culture treatment were measured in only one of the triplicate bioreactors and are thus unreplicated. In (b) and (c), results labeled with different letters (A, B) are statistically different from each other (p<0.0001).
Figure 5
Figure 5. Properties of cartilage constructs produced using PGA–alginate scaffolds cultured in bioreactors using a constant flow rate of 0.2 mL min−1 (high flow rate), a gradually increasing flow rate of 0.075–0.2 mL min−1 (gradual increase in flow rate), or scaffold pre-culture in T-flasks for 5 days prior to bioreactor culture at a constant flow rate of 0.2 mL min−1 (5-day pre-culture).
(a) Construct wet weight; and (b) number of cells. The scaffolds were seeded using 20×106 cells and cultured for a total of 5 weeks after seeding. The error bars represent standard errors from triplicate bioreactors.
Figure 6
Figure 6. Biochemical properties of cartilage constructs produced using PGA–alginate scaffolds cultured in bioreactors using a constant flow rate of 0.2 mL min−1 (high flow rate), a gradually increasing flow rate of 0.075–0.2 mL min−1 (gradual increase in flow rate), or scaffold pre-culture in T-flasks for 5 days prior to bioreactor culture at a constant flow rate of 0.2 mL min−1 (5-day pre-culture).
(a) GAG concentration; (b) total collagen concentration; (c) collagen type II concentration; and (d) collagen type II as a percentage of total collagen. The scaffolds were seeded using 20×106 cells and cultured for a total of 5 weeks after seeding. The error bars represent standard errors from triplicate bioreactors. Results labeled with different letters (A, B) are statistically different from each other (p<0.05).
Figure 7
Figure 7. Histological appearance of constructs produced using PGA–alginate scaffolds cultured in bioreactors for 5 weeks at a constant high flow rate of 0.2 mL min−1 (a, c, e) or a gradually increasing flow rate of 0.075–0.2 mL min−1 (b, d, f). The scaffolds were seeded using 20×106 cells.
Construct cross-sections show: (a, b) pink–red staining for GAG, blue staining for collagen, and dark blue–purple staining for cells; (c, d) immunostaining (brown) for collagen type I; and (e, f) immunostaining (brown) for collagen type II.
Figure 8
Figure 8. GAG release into the medium and retention in the constructs for PGA–alginate scaffolds cultured in bioreactors operated using a constant flow rate of 0.2 mL min−1 (high flow rate, •), a gradually increasing flow rate of 0.075–0.2 mL min−1 (gradual increase in flow rate, □), or scaffold pre-culture in T-flasks for 5 days prior to bioreactor culture at a constant flow rate of 0.2 mL min−1 (5-day pre-culture, ▪).
(a) Cumulative amount of GAG released into the medium; (b) overall specific rate of GAG release (mg per day per mg of GAG in the constructs at harvest); and (c) percentage of total GAG (construct+medium) retained in the constructs. The scaffolds were seeded using 20×106 cells and cultured for a total of 5 weeks after seeding. The error bars represent standard errors from triplicate bioreactors. Medium GAG data for the 5-day pre-culture treatment were measured in only one of the triplicate bioreactors and are thus unreplicated.
Figure 9
Figure 9. Analysis of proteoglycan size and integrity: (a and d) results from electrophoresis on composite acrylamide–agarose gels; (b and c) results from Western blots probed using monoclonal antibody specific to the hyaluronic-acid-binding region of human aggrecan.
The back-up nitrocellulose membrane for capture of smaller-sized molecules is shown in (b); the primary membrane showing larger-sized molecules is shown in (c). Lane 1 – aggrecan from bovine cartilage; Lane 2 – chondroitin sulphate from shark cartilage; Lane 3 – a 1∶1 w/w mixture of bovine aggrecan and chondroitin sulphate; Lane 4 – a 1∶1 w/w mixture of bovine aggrecan and proteoglycans isolated from human fetal cartilage; Lane 5 – proteoglycans isolated from human fetal cartilage; Lanes 6 and 7 –proteoglycans isolated from tissue-engineered cartilage; Lane 8 – spent medium from bioreactor culture of tissue-engineered cartilage; Lane 9 – bovine aggrecan digested with papain; Lane 10 – aggrecan from bovine cartilage; Lane 11 – proteoglycans isolated from human fetal cartilage; Lanes 12, 13 and 14 – spent medium from bioreactor culture of tissue-engineered cartilage. The sample in Lane 12 was diluted and treated with 6 M urea; the sample in Lane 14 was treated with hyaluronidase.

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