Optimizing nutrient channel spacing and revisiting TGF-beta in large engineered cartilage constructs

Abstract

Cartilage tissue engineering is a promising approach to treat osteoarthritis. However, current techniques produce tissues too small for clinical relevance. Increasingly close-packed channels have helped overcome nutrient transport limitations in centimeter-sized chondrocyte-agarose constructs, yet optimal channel spacings to recapitulate native cartilage compositional and mechanical properties in constructs this large have not been identified. Transient active TGF-β treatment consistently reproduces native compressive Young’s modulus (EY) and glycosaminoglycan (GAG) content in constructs, but standard dosages of 10 ng/mL exacerbate matrix heterogeneity. To ultimately produce articular layer-sized constructs, we must first optimize channel spacing and investigate the role of TGF-β in the utility of channels. We cultured ∅10 mm constructs with 0, 12, 19, or 27 nutrient channels (∅1 mm) for 6-8 weeks with 0, 1, or 10 ng/mL TGF-β; subsequently we analyzed them mechanically, biochemically, and histologically. Constructs with 12 or 19 channels grew the most favorably, reaching EY = 344 ± 113 kPa and GAG and collagen contents of 10.8% ± 1.2% and 2.2% ± 0.2% of construct wet weight, respectively. Constructs with 27 channels had significantly less deposited GAG than other groups. Channeled constructs given 1 or 10 ng/mL TGF-β developed similar properties. Without TGF-β, constructs with 0 or 12 channels exhibited properties that were indistinguishable, and lower than TGF-β-supplemented constructs. Taken together, these results emphasize that nutrient channels are effective only in the presence of TGF-β, and indicate that spacings equivalent to 12 channels in ∅10 mm constructs can be employed in articular-layersized constructs with reduced dosages of TGF-β.

Enhanced nutrient transport improves the depth-dependent properties of tri-layered engineered cartilage constructs with zonal co-culture of chondrocytes and MSCs

“…While channels appear promising during short term culture, we and others have shown that macro-channels can become filled with newly formed tissue early in culture (Supplementary 2). This can be addressed to some extent by ‘re-punching’ the closed channels [21], though this requires an additional handling step during culture. To overcome this limitation, in this study we included a porous HF or a HF strung with a cotton thread to maintain channel patency and improve nutrient/waste transport into the core of the construct.…”

“…This again suggests that the edge of the construct develops the highest properties and dominates both bulk and local mechanical response in constructs of this size. This may in the future be addressed by adding additional nutrient channels [21] and/or extending the culture duration and system to examine larger constructs where the edges are further removed.…”

Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-β signaling

“… 4 In juvenile bovine chondrocytes, brief, rather than sustained, exposure to TGF-β at concentrations lower than the typically applied supraphysiological dose of 10 ng/mL was sufficient for robust cartilage tissue formation. 3 , 5 , 6 Other studies that used integrated experimental and computational approaches have shown that TGF-β stimulation can induce short-term graded responses ( Smad7 gene expression) that evolved into long-term switch-like responses (growth inhibition). 7 …”

Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions

“…Introducing channels into cell laden hydrogels is a common strategy to facilitate enhanced nutrient transfer while engineering large cartilage tissues. 19 , 21 , 33 – 36 Alginate was used as the hydrogel material as it has been shown to support robust MSC chondrogenesis and supports a rounded chondrocyte-like cell morphology. 37 – 39 …”