Combining TGF-β1 and Mechanical Anchoring to Enhance Collagen Fiber Formation and Alignment in Tissue-Engineered...

Recapitulating the collagen fiber structure of native menisci is one of the major challenges in the development of tissue-engineered menisci. Native collagen fibers are developed by the complex interplay of biochemical and biomechanical signals. In this study, we optimized glucose and transforming growth factor-β1 (TGF-β1) concentrations in combination with mechanical anchoring to balance contributions of proteoglycan synthesis and contractile behavior in collagen fiber assembly. Glucose had a profound effect on the final dimensions of collagen-based constructs. TGF-β1 influenced construct contraction rate and glycosaminoglycan (GAG) production with two half-maximal effective concentration (EC₅₀) ranges, which are 0.23 to 0.28 and 0.53 to 1.71 ng/mL, respectively. At concentrations less than the EC₅₀, for the GAG production and contraction rate, TGF-β1 treatment resulted in less organized collagen fibers. At concentrations greater than the EC₅₀, TGF-β1 led to dense, disorganized collagen fibers. Between the two EC₅₀ values, collagen fiber diameter and length increased. The effects of TGF-β1 on fiber development were enhanced by mechanical anchoring, leading to peaks in fiber diameter, length, and alignment index. Fiber diameter and length increased from 7.9 ± 1.4 and 148.7 ± 16.4 to 17.5 ± 2.1 and 262.0 ± 13.0 μm, respectively. The alignment index reached 1.31, comparable to that of native tissue, 1.40. These enhancements in fiber architecture resulted in significant increases in tensile modulus and ultimate tensile stress (UTS) by 1.6- and 1.4-fold. Correlation analysis showed that tensile modulus and UTS strongly correlated with collagen fiber length, diameter, and alignment, while compressive modulus correlated with GAG content. These outcomes highlight the need for optimization of both biochemical and biomechanical cues in the culture environment for enhancing fiber development within tissue-engineered constructs.