School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia.
PLoS One. 2011;6(8):e23119. doi: 10.1371/journal.pone.0023119. Epub 2011 Aug 15.
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.
组织工程软骨的生产涉及关键成分如糖胺聚糖（GAG）和 II 型胶原的合成和积累，以形成不溶性细胞外基质（ECM）。在软骨培养过程中，大分子成分从新生组织释放到培养基中，这代表了生物合成资源的巨大浪费。这项工作旨在开发提高软骨构建体中 ECM 保留率的策略，从而提高生物反应器中产生的工程组织的质量。将人软骨细胞接种到聚乙醇酸（PGA）支架中，在灌注生物反应器中培养长达 5 周。对构建体和培养基中蛋白聚糖的大小和完整性进行分析表明，完整大小的聚集蛋白聚糖正在从组织中被剥离，而没有发生蛋白水解降解。在生物反应器中应用低（0.075 mL min(-1)) 和逐渐增加（0.075-0.2 mL min(-1)) 的培养基流速导致生成更大的构建体，ECM 中保留的 GAG 百分比增加了 4.0-4.4 倍，组织中 GAG 浓度增加了 4.8-5.2 倍与 0.2 mL min(-1)的操作相比。在生物反应器培养之前，将接种的支架在培养瓶中预培养 5 天，也可以改善 GAG 的保留。相比之下，用藻酸盐水凝胶灌注的 PGA 支架中的 GAG 保留率与培养基流速或预培养处理没有显著差异。这项工作表明，使用专门针对和改善 ECM 保留率的支架和生物反应器培养策略，可以显著提高软骨的质量。
