Physiologic Doses of Transforming Growth Factor-β Improve the Composition of Engineered Articular Cartilage

Conventionally, for cartilage tissue engineering applications, transforming growth factor beta (TGF-β) is administered at doses that are several orders of magnitude higher than those present during native cartilage development. While these doses accelerate extracellular matrix (ECM) biosynthesis, they may also contribute to features detrimental to hyaline cartilage function, including tissue swelling, type I collagen (COL-I) deposition, cellular hypertrophy, and cellular hyperplasia. In contrast, during native cartilage development, chondrocytes are exposed to moderate TGF-β levels, which serve to promote strong biosynthetic enhancements while mitigating risks of pathology associated with TGF-β excesses. Here, we examine the hypothesis that physiologic doses of TGF-β can yield neocartilage with a more hyaline cartilage-like composition and structure relative to conventionally administered supraphysiologic doses. This hypothesis was examined on a model system of reduced-size constructs (∅2 × 2 mm or ∅3 × 2 mm) comprised of bovine chondrocytes encapsulated in agarose, which exhibit mitigated TGF-β spatial gradients allowing for an evaluation of the intrinsic effect of TGF-β doses on tissue development. Reduced-size (∅2 × 2 mm or ∅3 × 2 mm) and conventional-size constructs (∅4-∅6 mm × 2 mm) were subjected to a range of physiologic (0.1, 0.3, 1 ng/mL) and supraphysiologic (3, 10 ng/mL) TGF-β doses. At day 56, the physiologic 0.3 ng/mL dose yielded reduced-size constructs with native cartilage-matched Young's modulus (E_Y) (630 ± 58 kPa) and sulfated glycosaminoglycan (sGAG) content (5.9 ± 0.6%) while significantly increasing the sGAG-to-collagen ratio, leading to significantly reduced tissue swelling relative to constructs exposed to the supraphysiologic 10 ng/mL TGF-β dose. Furthermore, reduced-size constructs exposed to the 0.3 ng/mL dose exhibited a significant reduction in fibrocartilage-associated COL-I and a 77% reduction in the fraction of chondrocytes present in a clustered morphology, relative to the supraphysiologic 10 ng/mL dose (p < 0.001). E_Y was significantly lower for conventional-size constructs exposed to physiologic doses due to TGF-β transport limitations in these larger tissues (p < 0.001). Overall, physiologic TGF-β appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating features detrimental to hyaline cartilage function. While reduced-size constructs are not suitable for the repair of clinical-size cartilage lesions, insights from this work can inform TGF-β dosing requirements for emerging scaffold release or nutrient channel delivery platforms capable of achieving uniform delivery of physiologic TGF-β doses to larger constructs required for clinical cartilage repair.