Enthesis maturation in engineered ligaments is differentially driven by loads that mimic slow growth elongation and rapid cyclic muscle movement.

Entheses are complex attachments that translate load between elastic-ligaments and stiff-bone via organizational and compositional gradients. Neither natural healing, repair, nor engineered replacements restore these gradients, contributing to high re-tear rates. Previously, we developed a culture system which guides ligament fibroblasts in high-density collagen gels to develop early postnatal-like entheses, however further maturation is needed. Mechanical cues, including slow growth elongation and cyclic muscle activity, are critical to enthesis development in vivo but these cues have not been widely explored in engineered entheses and their individual contribution to maturation is largely unknown. Our objective here was to investigate how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, individually drive enthesis maturation in our system so to shed light on the cues governing enthesis development, while further developing our tissue engineered replacements. Interestingly, we found these loads differentially drive organizational maturation, with slow stretch driving improvements in the interface/enthesis region, and cyclic load improving the ligament region. However, despite differentially affecting organization, both loads produced improvements to interface mechanics and zonal composition. This study provides insight into how mechanical cues differentially affect enthesis development, while producing some of the most organized engineered enthesis to date. STATEMENT OF SIGNIFICANCE: Entheses attach ligaments to bone and are critical to load transfer; however, entheses do not regenerate with repair or replacement, contributing to high re-tear rates. Mechanical cues are critical to enthesis development in vivo but their individual contribution to maturation is largely unknown and they have not been widely explored in engineered replacements. Here, using a novel culture system, we provide new insight into how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, differentially affect enthesis maturation in engineered ligament-to-bone tissues, ultimately producing some of the most organized entheses to date. This system is a promising platform to explore cues regulating enthesis formation so to produce functional engineered replacements and better drive regeneration following repair.

Temporal application of lysyl oxidase during hierarchical collagen fiber formation differentially effects tissue mechanics.

The primary source of strength in menisci, tendons, and ligaments are hierarchical collagen fibers; however, these fibers are not regenerated after injury nor in engineered replacements, resulting in limited repair options. Collagen strength is reliant on fiber alignment, density, diameter, and crosslinking. Recently, we developed a culture system which guides cells in high-density collagen gels to develop native-like hierarchically organized collagen fibers, which match native fiber alignment and diameters by 6 weeks. However, tensile moduli plateau at 1MPa, suggesting crosslinking may be lacking. Collagen crosslinking is regulated by lysyl oxidase (LOX) which forms immature crosslinks that condense into mature trivalent crosslinks. Trivalent crosslinks are thought to be the primarily source of strength in fibers, but it's not well understood how they form. The objective of this study was to evaluate the effect of exogenous LOX in our culture system at different stages of hierarchical fiber formation to produce stronger replacements and to better understand factors affecting crosslink maturation. We found treatment with LOX isoform LOXL2 did not restrict hierarchical fiber formation, with constructs still forming aligned collagen fibrils by 2 weeks, larger fibers by 4 weeks, and early fascicles by 6 weeks. However, LOXL2 treatment did significantly increase mature pyridinium (PYD) crosslink accumulation and tissue mechanics, with timing of LOXL2 supplementation during fiber formation having a significant effect. Overall, we found one week of LOXL2 supplementation at 4 weeks produced constructs with native-like fiber organization, increased PYD accumulation, and increased mechanics, ultimately matching the tensile modulus of immature bovine menisci. STATEMENT OF SIGNIFICANCE: Collagen fibers are the primary source of strength and function in connective tissues throughout the body, however it remains a challenge to develop these fibers in engineered replacements, greatly reducing treatment options. Here we demonstrate lysyl oxidase like 2 (LOXL2) can be used to significantly improve the mechanics of tissue engineered constructs, but timing of application is important and will most likely depend on degree of collagen organization or maturation. Currently there is limited understanding of how collagen crosslinking is regulated, and this system is a promising platform to further investigate cellular regulation of LOX crosslinking. Understanding the mechanism that regulates LOX production and activity is needed to ultimately regenerate functional repair or replacements for connective tissues throughout the body.

Driving native-like zonal enthesis formation in engineered ligaments using mechanical boundary conditions and ß-tricalcium phosphate.

Fibrocartilaginous entheses are structurally complex tissues that translate load from elastic ligaments to stiff bone via complex zonal gradients in the organization, mineralization, and cell phenotype. Currently, these complex gradients necessary for long-term mechanical function are not recreated in soft tissue-to-bone healing or engineered replacements, contributing to high failure rates. Previously, we developed a culture system that guides ligament fibroblasts to develop aligned native-sized collagen fibers using high-density collagen gels and mechanical boundary conditions. These constructs are promising 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 (ßTCP), 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. Ultimately, by 6 weeks of culture these constructs had similar organization and composition as immature bovine entheses. Further, ßTCP applied under the clamp enhanced maturation of these entheses, leading to significantly increased tensile moduli, and zonal GAG accumulation, ALP activity, and calcium-phosphate accumulation, 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. STATEMENT OF SIGNIFICANCE: Ligaments are attached to bone via entheses. Entheses are complex tissues with gradients in organization, composition, and cell phenotype. Entheses are necessary for proper transfer of load from ligament-to-bone, but currently are not restored with healing or replacements. Here, we provide new insight into how tensile-compressive boundary conditions and ßTCP drive zonal gradients in collagen organization, mineralization, and matrix composition, producing tissues similar to immature ligament-to-bone attachments. Collectively, this culture system uses a bottom-up approach with mechanical and biochemical cues to produce engineered replacements which closely mirror postnatal enthesis development. This culture system is a promising platform to better understanding the cues that regulate enthesis formation so to better drive enthesis regeneration following graft repair and in engineered replacements.

An inducible model for unraveling the effects of advanced glycation end-product accumulation in aging connective tissues.

PURPOSE: In connective tissues there is a clear link between increasing age and degeneration. Advanced glycation end-products (AGEs) are believed to play a central role. AGEs are sugar-induced non-enzymatic crosslinks which accumulate in collagen with age and diabetes, altering tissue mechanics and cellular function. Despite ample correlative evidence linking collagen glycation to tissue degeneration, little is known how AGEs impact cell-matrix interactions, limiting therapeutic options. One reason for this limited understanding is that AGEs are typically induced using high concentrations of ribose which decrease cell viability, making it impossible to investigate cell-matrix interactions. The objective of this study was to develop a system to trigger AGE accumulation while maintaining cell viability. MATERIALS AND METHODS: Using cell-seeded high density collagen gels, we investigated the effect of two systems for AGE induction, ribose at low concentrations (30, 100, and 200 mM) over 15 days of culture and riboflavin (0.25 and 0.75 mM) induced with blue light for 40 seconds (riboflavin-465 nm). RESULTS: We found ribose and riboflavin-465 nm treatment produces fluorescent AGE quantities which match and/or exceed human fluorescent AGE levels for various tissues, ages, and diseases, without affecting cell viability or metabolism. Interestingly, a 40 second treatment of riboflavin-465 nm produced similar levels of fluorescent AGEs as 3 days of 100 mM ribose treatment. CONCLUSIONS: Riboflavin-465 nm is a promising method to trigger AGEs on demand in vivo or in vitro without impacting cell viability and offers potential for unraveling the mechanism of AGEs in age and diabetes related tissue damage.