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

Conventionally, for cartilage tissue engineering applications, transforming growth factor beta (TGF-ß) 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-ß levels, which serve to promote strong biosynthetic enhancements while mitigating risks of pathology associated with TGF-ß excesses. Here, we examine the hypothesis that physiologic doses of TGF-ß 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-ß spatial gradients allowing for an evaluation of the intrinsic effect of TGF-ß 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-ß doses. At day 56, the physiologic 0.3 ng/mL dose yielded reduced-size constructs with native cartilage-matched Young's modulus (EY) (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-ß 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). EY was significantly lower for conventional-size constructs exposed to physiologic doses due to TGF-ß transport limitations in these larger tissues (p < 0.001). Overall, physiologic TGF-ß 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-ß dosing requirements for emerging scaffold release or nutrient channel delivery platforms capable of achieving uniform delivery of physiologic TGF-ß doses to larger constructs required for clinical cartilage repair.

Physiologic Doses of TGF-ß Improve the Composition of Engineered Articular Cartilage.

For cartilage regeneration applications, transforming growth factor beta (TGF-ß) is conventionally administered at highly supraphysiologic doses (10-10,000 ng/mL) in an attempt to cue cells to fabricate neocartilage that matches the composition, structure, and functional properties of native hyaline cartilage. While supraphysiologic doses enhance ECM biosynthesis, they are also associated with inducing detrimental tissue features, such as fibrocartilage matrix deposition, pathologic-like chondrocyte clustering, and tissue swelling. Here we investigate the hypothesis that moderated TGF-ß doses (0.1-1 ng/mL), akin to those present during physiological cartilage development, can improve neocartilage composition. Variable doses of media-supplemented TGF-ß were administered to a model system of reduced-size cylindrical constructs (Ø2-Ø3 mm), which mitigate the TGF-ß spatial gradients observed in conventional-size constructs (Ø4-Ø6 mm), allowing for a novel assessment of the intrinsic effect of TGF-ß doses on macroscale neocartilage properties and composition. The administration of physiologic TGF-ß to reduced-size constructs yields neocartilage with native-matched sGAG content and mechanical properties while providing a more hyaline cartilage-like composition, marked by: 1) reduced fibrocartilage-associated type I collagen, 2) 77% reduction in the fraction of cells present in a clustered morphology, and 3) 45% reduction in the degree of tissue swelling. Physiologic TGF-ß appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating hyaline cartilage compositional deficits. These results can guide the development of novel physiologic TGF-ß-delivering scaffolds to improve the regeneration clinical-sized neocartilage tissues.

Physiologic Medium Maintains the Homeostasis of Immature Bovine Articular Cartilage Explants in Long-Term Culture.

The ability to maintain living articular cartilage tissue in long-term culture can serve as a valuable analytical research tool, allowing for direct examination of mechanical or chemical perturbations on tissue behavior. A fundamental challenge for this technique is the recreation of the salient environmental conditions of the synovial joint in culture that are required to maintain native cartilage homeostasis. Interestingly, conventional media formulations used in explanted cartilage tissue culture investigations often consist of levels of metabolic mediators that deviate greatly from their concentrations in synovial fluid (SF). Here, we hypothesize that the utilization of a culture medium consisting of near-physiologic levels of several highly influential metabolic mediators (glucose, amino acids, cortisol, insulin, and ascorbic acid) will maintain the homeostasis of cartilage explants as assessed by their mechanical properties and extracellular matrix (ECM) contents. Results demonstrate that the aforementioned mediators have a strong effect on the mechanical and biochemical stability of skeletally immature bovine cartilage explants. Most notably, (1) in the absence of cortisol, explants exhibit extensive swelling and tissue softening and (2) in the presence of supraphysiologic levels of anabolic mediators (glucose, amino acids, insulin), explants exhibit increased matrix accumulation and tissue stiffening. In contrast, the administration of physiologic levels of these mediators (as present in native SF) greatly improves the stability of live cartilage explants over one month of culture. These results may have broad applicability for articular cartilage and other musculoskeletal tissue research, setting the foundation for important culture formulations required for examinations into tissue behavior.