Ng K W, Kugler L E, Doty S B, Ateshian G A, Hung C T
Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA.
Osteoarthritis Cartilage. 2009 Feb;17(2):220-7. doi: 10.1016/j.joca.2008.06.013. Epub 2008 Sep 17.
It was hypothesized that controlled, scaffold removal in engineered cartilage constructs would improve their collagen content and mechanical properties over time in culture.
Preliminary experiments characterized the effects of agarase on cell-free agarose disks and cartilage explants. Immature bovine chondrocytes were encapsulated in agarose, cultured to day 42, and incubated with 100 units/mL agarase for 48 h. After treatment, constructs were cultured to day 91. The compressive Young's modulus and dynamic modulus of the constructs were determined every 2 weeks and immediately after agarase treatment. Post-mechanical testing, constructs were processed for biochemistry and histology.
Agarase treatment on explants had no detrimental effect on the cartilage matrix. Treatment applied to engineered constructs on day 42 did not affect DNA or collagen content. Agarase treatment decreased tissue GAG content (via GAG loss to the media) and Young's modulus, both of which recovered to control values over time in culture. By day 91 agarase-treated constructs possessed approximately 25% more DNA, approximately 60% more collagen, and approximately 40% higher dynamic modulus compared to untreated controls.
Scaffold degradation increased construct collagen content and dynamic mechanical properties, affirming the experimental hypothesis. The mechanism may lie in increased nutrient transport, increased space for collagen fibril formation, and cellular response to the loss of GAG with agarase treatment. The results highlight the role of the scaffold in retaining synthesized matrix during early and late tissue formation. This work also shows promise in developing an engineered tissue that may be completely free of scaffold material for clinical implantation.
提出的假设是，在培养过程中，对工程化软骨构建体进行可控的支架去除会随着时间的推移改善其胶原蛋白含量和力学性能。
初步实验表征了琼脂酶对无细胞琼脂糖圆盘和软骨外植体的影响。将未成熟的牛软骨细胞封装在琼脂糖中，培养至第42天，并用100单位/毫升的琼脂酶孵育48小时。处理后，将构建体培养至第91天。每2周以及在琼脂酶处理后立即测定构建体的压缩杨氏模量和动态模量。机械测试后，对构建体进行生物化学和组织学处理。
对软骨外植体进行琼脂酶处理对软骨基质没有有害影响。在第42天对工程化构建体进行的处理不影响DNA或胶原蛋白含量。琼脂酶处理降低了组织糖胺聚糖含量（通过糖胺聚糖流失到培养基中）和杨氏模量，两者在培养过程中均随时间恢复到对照值。与未处理的对照相比，到第91天时，经琼脂酶处理的构建体的DNA含量增加了约25%，胶原蛋白含量增加了约60%，动态模量提高了约40%。
支架降解增加了构建体的胶原蛋白含量和动态力学性能，证实了实验假设。其机制可能在于营养物质运输增加、胶原蛋白原纤维形成空间增大以及细胞对琼脂酶处理导致的糖胺聚糖流失的反应。结果突出了支架在组织早期和晚期形成过程中保留合成基质的作用。这项工作在开发一种可能完全不含支架材料用于临床植入的工程组织方面也显示出前景。
