Physioxia stimulates extra-cellular matrix deposition and increases mechanical properties of human chondrocyte-derived tissue-engineered cartilage

Abstract

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at one- and two-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio.SummaryTissue-engineered cartilage formed from human articular chondrocytes produces thicker, stiffer, more extracellular-matrix rich cartilage tissue when grown under physiological (5%) vs. atmospheric oxygen (20%) tension.

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“…The results show a significantly increased cell expansion rate between all three test conditions (SCM, SCM + bFGF, bFGF) and the control (TCP) (p < 0.0001), sustained throughout the four passages. This is consistent with our previous work that has shown enhanced growth of chondrocytes with the use of SCM compared to TCP (Kean et al, 2019;Dennis et al, 2020). In addition, chondrocytes cultured in SCM + bFGF showed a significantly higher expansion rate than in bFGF alone in all four passages, indicating that there is some additional benefit to including both components.…”

“…This increase in types I and X collagen and a decrease in type II were most apparent in the TCP group. In a previous work, it was observed that the type I collagen in our tissue-engineered cartilage reduced with increased culture time to 2 months, something we did not investigate here (Dennis et al, 2020).…”

“…At the end of the 21-day culture period, cartilage aggregates were assessed for wet weight, DNA, and glycosaminoglycan and hydroxyproline content as previously described (Dennis et al, 2020). Briefly, the aggregates were digested with papain, and then the digests were split between DNA/glycosaminoglycan (GAG) assay and hydroxyproline (HDP) assays.…”

“…Zhou et al (2004) have also previously demonstrated via a modeling approach that the rate of oxygen consumption in the articular cartilage fell in an oxygen tension-dependent manner once the tissue oxygen tension fell below 5%. The use of oxygen tension at 5% has been shown by multiple groups, including our own, to be beneficial for chondrocyte culture (Lafont et al, 2008;Das et al, 2010;Kean and Dennis, 2015;Anderson et al, 2018;Dennis et al, 2020). Oxygen tension of 5% was, therefore, chosen because of this decreased level within the range of oxygen tensions reported as physiological.…”

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“…MSCs have also shown increased ECM production when cocultured with chondrocytes [203]. Both MSCs [203] and primary articular chondrocytes [54] have a high level of donor to donor variability. This variability is a disadvantage in using cells from any primary source.…”