RESUMEN
OBJECTIVE: It was hypothesized that controlled, scaffold removal in engineered cartilage constructs would improve their collagen content and mechanical properties over time in culture. DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Cartílago Articular/fisiología , Colágeno/metabolismo , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/metabolismo , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/ultraestructura , Bovinos , Condrocitos/citología , Fuerza Compresiva/fisiología , Glicósido Hidrolasas/farmacología , Ensayo de Materiales/métodos , Microscopía Electrónica de Rastreo , Proteoglicanos/metabolismo , Sefarosa/metabolismoRESUMEN
Increased amino acid supplementation (0.5 x, 1.0 x, and 5.0 x recommended concentrations or additional proline) was hypothesized to increase the collagen content in engineered cartilage. No significant differences were found between groups in matrix content or dynamic modulus. Control constructs possessed the highest compressive Young's modulus on day 42. On day 42, compared to controls, decreased type II collagen was found with 0.5 x, 1.0 x, and 5.0 x supplementation and significantly increased DNA content found in 1.0 x and 5.0 x. No effects were observed on these measures with added proline. These results lead us to reject our hypothesis and indicate that the low collagen synthesis in engineered cartilage is not due to a limited supply of amino acids in media but may require a further stimulatory signal. The results of this study also highlight the impact that culture environment can play on the development of engineered cartilage.