Abstract
In cartilage tissue engineering, active TGF-β is conventionally supplemented in culture medium at highly supraphysiologic doses to accelerate neocartilage development. While this approach enhances cartilage extracellular matrix (ECM) biosynthesis, it further promotes tissue features detrimental to hyaline cartilage function, including the induction of tissue swelling, hyperplasia, hypertrophy, and ECM heterogeneities. In contrast, during native cartilage development, chondrocytes are surrounded by TGF-β configured in a latent complex (LTGF-β), which undergoes cell-mediated activation, giving rise to moderated, physiologic dosing regimens that enhance ECM biosynthesis while avoiding detrimental features associated with TGF-β excesses. Here, we explore a bio-inspired strategy, consisting of LTGF-β-conjugated scaffolds, providing TGF-β exposure regimens that are moderated and uniformly administered throughout the construct. Specifically, we evaluate the performance of LTGF-β scaffolds to improve neocartilage development with bovine chondrocyte-seeded agarose constructs compared to outcomes from active TGF-β media supplementation (MS) at a physiologic 0.3 ng/mL dose (MS-0.3), supraphysiologic 10 ng/mL dose (MS-10), or TGF-β free. For small-size constructs (∅3×2 mm), LTGF-β scaffolds yield neocartilage that achieves native-matched mechanical properties (800-925 kPa) and sGAG content (6.6%-7.1%), while providing a cell morphology and collagen distribution more reminiscent of hyaline cartilage. LTGF-β scaffolds further afford an optimal chondrogenic phenotype, marked by a 12-to 28-fold reduction of COL-I expression relative to TGF-β-free and a 7-to 17-fold reduction of COL-X expression relative to MS-10. Further, for large-size constructs, which approach the dimensions needed for clinical cartilage repair, LTGF-β scaffolds significantly reduce mechanical and biochemical heterogeneities relative to MS-0.3 and MS-10. Overall, the use of LTGF-β scaffolds improves the composition, structure, material properties, and cell phenotype of neocartilage.