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 (βTCP), 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, βTCP 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.

Advanced strategies for constructing interfacial tissues of bone and tendon/ligament

“…A suitable biological enthesis scaffold should have the following properties: (a) a biomimetic structure, (b) mechanically biomimetic properties, (c) mouldability, (d) biodegradability, and (e) biocompatibility (should not be cytotoxic or inflammatory). 3,42,116–118 These requirements markedly limit the number of materials that can be used as biological scaffolds in enthesis TE. Enthesis scaffolds can be natural or artificial.…”

“…[110][111][112] However, the development of enthesis TE is not as advanced and the successful reconstruction of enthesis tissue has not yet been achieved. 3,26,[113][114][115] Studies on the use of TE in enthesis repair involve four strategies, the biological scaffold, cell, growth factor, and biophysical modulation strategies. Of these, the biological scaffold strategy has attracted the most interest (Table 1).…”

“…1,2 Enthesis also supports the mechanical transmission of tensile stress and force from the main body of the tendon/ligament to the bone surface. 3–5 Because of its location between the tendon/ligament and the bone, the enthesis is frequently damaged during avulsion fracture and ankylosing spondylitis. 6,7 The incidence rate of enthesis orthopedic injury has increased markedly in recent years, and each year, around 30 million people undergo enthesis reconstruction in the United States and Europe, which costs over 163 billion dollars.…”

“…The advanced methods that are currently used in enthesis tissue engineering can be divided into the biological scaffold strategy, the cell strategy, the growth factor strategy, and the biophysical modulation strategy (Figure 1). 3,[26][27][28] These strategies can be used alone or in combination to promote enthesis regeneration. The biggest challenge in enthesis regeneration is the lack of effective ways of generating the typical four-layered histological structure at the interface between the bone and the broken enthesis tissue.…”

Mechanical Evaluation of Hydrogel-Elastomer Interfaces Generated Through Thiol-Ene Coupling

“…This ranges from advanced in vitro models making use of hydrophobic elastomeric structures to compartmentalise and mechanically stimulate soft cell-encapsulating domains [32][33][34][35][36] to the engineering of tissue engineering scaffolds [37][38][39] . Such materials and associated microfabricated platforms are attractive for the study, control and repair of soft-hard tissue interfaces 40,41 . Although the engineering of thiol-ene based soft hydrogel enabling cell encapsulation, such as gelatin methacrylate systems, has enabled their integration within a broad range of polyesters [42][43][44][45] or silicones 46,47 , the study of their mechanical integration and adhesion to corresponding elastomers has received relatively little attention.…”