Comparative analysis of different macromolecular crowding agents in human tenocyte cultures

• κλ CR and λ CR induced the highest collagen type I deposition. • Polydispersity index had the highest correlation to collagen type I deposition. • Gene expression analysis revealed a MMC agent-dependent cell response. • This work advocates the use of κλ CR and λ CR in tendon engineering. Tendon engineered substitutes remain clinically unavailable, largely due to the need for prolonged culture periods to allow for sufficient extracellular matrix deposition. Although macromolecular crowding allows for accelerated extracellular matrix deposition, the optimal macromolecular crowding agent in human tenocyte cultures is still unknown. Here, we measured the physicochemical properties of six macromolecules (κλ carrageenan, λ carrageenan, polysucrose, polyacrylic acid, hyaluronic acid and polyvinylpyrrolidone) and assessed their effect in human tenocyte basic function, collagen type I deposition and gene expression. The κλ carrageenan exhibited the highest polydispersity index. None of the macromolecules impaired cell viability and metabolic activity. The κλ carrageenan and the λ carrageenan significantly enhanced collagen type I deposition. A macromolecular crowding agent dependent gene expression was observed, with the κλ carrageenan appearing to induce a more mature phenotype. This study provides a framework for selecting an appropriate macromolecular crowding agent to enhance and accelerate extracellular matrix deposition, whilst maintaining acceptable cell function. Identification of optimal macromolecular crowding agent in human tenocyte cultures.