Nims Robert J, Cigan Alexander D, Durney Krista M, Jones Brian K, O'Neill John D, Law Wing-Sum A, Vunjak-Novakovic Gordana, Hung Clark T, Ateshian Gerard A
1 Department of Biomedical Engineering, Columbia University , New York, New York.
2 Department of Mechanical Engineering, Columbia University , New York, New York.
Tissue Eng Part A. 2017 Aug;23(15-16):847-858. doi: 10.1089/ten.TEA.2016.0467. Epub 2017 Mar 27.
When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel "cage" growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-β before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.
在充足营养供应的条件下培养时，工程化软骨会迅速合成蛋白聚糖，产生渗透压肿胀压力，这种压力会破坏未成熟的胶原蛋白，并阻碍强健胶原蛋白框架的形成，而强健的胶原蛋白框架是天然软骨的一个标志。我们推测，机械限制蛋白聚糖诱导的组织肿胀将通过形成更稳定的胶原蛋白框架来增强构建体的功能特性。为了验证这一假设，我们开发了一种新型的“笼式”生长系统，以机械方式防止组织构建体肿胀，同时确保为生长中的构建体提供充足的营养供应。通过测试嵌入两种不同支架（琼脂糖和软骨衍生基质水凝胶（CDMH））中的构建体，来检验受限培养的有效性。构建体接种未成熟的牛软骨细胞，并在自由肿胀（FS）条件下用转化生长因子-β培养14天，然后在培养的剩余时间放入约束笼中。对照组在整个培养过程中均在FS条件下培养。在第14天放入笼子后，在笼子中培养的琼脂糖构建体不再膨胀，而FS构建体在培养112天后膨胀至其第0天重量的8倍。除了生长方面的物理差异外，到第56天时，笼养构建体的平衡模量（琼脂糖：639±179kPa，CDMH：608±2
