Driving Native-like Zonal Enthesis Formation in Engineered Ligaments Using Mechanical Boundary Conditions and β-Tricalcium Phosphate

Abstract

Fibrocartilaginous entheses are structurally complex tissues that translate load from elastic ligaments to stiff bone via complex zonal organization with gradients in organization, mineralization, and cell phenotype. Currently, these gradients, necessary for long-term mechanical function, are not recreated in soft tissue-to-bone healing or engineered replacements, leading to high failure rates. Previously, we developed a culture system which guides ligament fibroblasts to develop aligned native-sized collagen fibers using high density collagen gels and mechanical boundary conditions. These constructs hold great promise as ligament replacements, however functional ligament-to-bone attachments, or entheses, are required for long-term function in vivo. The objective of this study was to investigate the effect of compressive mechanical boundary conditions and the addition of beta tricalcium phosphate ({beta}TCP), a known osteoconductive agent, on the development of zonal ligament-to-bone entheses. We found that compressive boundary clamps, that restrict cellular contraction and produce a zonal tensile-compressive environment, guide ligament fibroblasts to produce 3 unique zones of collagen organization, and zonal accumulation of glycosaminoglycans (GAGs), type II and type X collagen by 6 weeks of culture, ultimately resulting in similar organization and composition as immature bovine entheses. Further, {beta}TCP under the clamp enhanced the maturation of these entheses, leading to increased GAG accumulation, sheet-like mineralization, and significantly improved tensile moduli, suggesting the initiation of endochondral ossification. This culture system produced some of the most organized entheses to date, closely mirroring early postnatal enthesis development, and provides an in vitro platform to better understand the cues that drive enthesis maturation in vivo.