Heterogeneous engineered cartilage growth results from gradients of media-supplemented active TGF-β and is ameliorated by the alternative supplementation of latent TGF-β

Abstract

Transforming growth factor beta (TGF-β) has become one of the most widely utilized mediators of engineered cartilage growth. It is typically exogenously supplemented in the culture medium in its active form, with the expectation that it will readily transport into tissue constructs through passive diffusion and influence cellular biosynthesis uniformly. The results of this investigation advance three novel concepts regarding the role of TGF-β in cartilage tissue engineering that have important implications for tissue development. First, through the experimental and computational analysis of TGF-β concentration distributions, we demonstrate that, contrary to conventional expectations, media-supplemented exogenous active TGF-β exhibits a pronounced concentration gradient in tissue constructs, resulting from a combination of high-affinity binding interactions and a high cellular internalization rate. These gradients are sustained throughout the entire culture duration, leading to highly heterogeneous tissue growth; biochemical and histological measurements support that while biochemical content is enhanced up to 4-fold at the construct periphery, enhancements are entirely absent beyond 1 mm from the construct surface. Second, construct-encapsulated chondrocytes continuously secrete large amounts of endogenous TGF-β in its latent form, a portion of which undergoes cell-mediated activation and enhances biosynthesis uniformly throughout the tissue. Finally, motivated by these prior insights, we demonstrate that the alternative supplementation of additional exogenous latent TGF-β enhances biosynthesis uniformly throughout tissue constructs, leading to enhanced but homogeneous tissue growth. This novel demonstration suggests that latent TGF-β supplementation may be utilized as an important tool for the translational engineering of large cartilage constructs that will be required to repair the large osteoarthritic defects observed clinically.

Dexamethasone Release from Within Engineered Cartilage as a Chondroprotective Strategy Against Interleukin-1α

“…86,87 The high binding affinity of active TGF-b3, an *25 kDa molecule (compared with 400 Da for dex), to agarose and cartilage matrix has also been implicated in contributing some part to the development of tissue heterogeneity. 88 The E Y values observed here are interestingly in tandem with cellular density and swelling ratio. With respect to cellularity, tissues that received no dex throughout the study (ULMS) proliferated to a cellular density *2.75-fold higher than those that did (ctrl, LMS) (Fig.…”

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties

“…Transient TGF-β treatment, which has previously shown successes with juvenile bovine constructs, was ineffective for hACs (Figure 3), similar to our previous findings with passaged adult canine chondrocytes (Ng et al, 2008b). However, ongoing research suggests that beneficial effects of active TGF-β are limited to constructs’ surfaces and that more sophisticated delivery strategies may be necessary for homogeneous tissue growth (Albro et al, 2013; Albro et al, 2016). In these early experiments with hACs in 3D culture our knowledge of optimal growth factor treatments is not yet complete, and we are hopeful that improved delivery of TGF-β and other factors will prove useful in encouraging homogeneous and sustained growth of hAC-laden constructs.…”

“…Agarose hydrogel scaffolds stabilize the chondrocyte phenotype (Benya and Shaffer, 1982; Buschmann et al, 1992; Mauck et al, 2000), and they promote deposition of proteoglycans that are most similar to those of native cartilage compared to other scaffold types (Mouw et al, 2005). Increasing initial cell seeding densities up to or above 60 × 10 6 cells/mL leads to concomitant increases in matrix synthesis, though this steepens nutrient gradients within the tissue and promotes heterogeneity (Albro et al, 2016; Bian et al, 2009a; Talukdar et al, 2011; Zhou et al, 2008). Tissue heterogeneity is mitigated by nutrient channels, which minimize nutrient diffusion distances and improve functional properties (Bian et al, 2009a; Buckley et al, 2009; Cigan et al, 2013b; Nims et al, 2016).…”

Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

“…Chondrogenesis of MSCs in vitro is defined by a temporal sequence of cellular events (3). We recently showed that isotropic chondrogenic induction of hMSCs in hydrogel formed cartilage with a deficient core (23). In other studies, isotropic hypertrophic induction of chondrogenically differentiated hMSCs resulted in uncontrolled mineralization (4,12).…”