Biology subjects

Puetzer, J. L.

Publications and source records attributed to Puetzer, J. L..

7 recordsLinked to original sources

bioRxiv2026

Stepped Cyclic Strain, that Increases or Decreases as Hierarchical Collagen Fibers Form, Does not Further Improve Maturation in Engineered Ligaments

The primary source of strength in ligaments and tendons are hierarchically organized collagen fibers. These fibers largely do not regenerate after injury, with repair, nor in engineered replacements, limiting treatment options. Previously, we developed a culture system which guides ACL fibroblasts in high-density collagen gels to form native-size hierarchical fibers over 6 weeks, and demonstrated that intermittent cyclic stretch further improves maturation. However, additional maturation is needed for clinical relevance. Interestingly, we found cyclic load affected cells differentially depending on the degree of organization, with 10% cyclic strain driving early improvements in unorganized gels and 5% strain being more beneficial later in culture once cells were on aligned fibers. Here, we explored whether a stepped cyclic load, that increased or decreased in strain magnitude as collagen fibers developed, further improved maturation. We hypothesized that progressively decreasing cyclic strain as organization increases would drive cells to produce more mature hierarchical fibers, resulting in stronger replacements. Controls had intermittent cyclic stretch at 0, 5, 7, or 10% strain throughout culture, while stepped load constructs were cyclically loaded with a strain that increased or decreased by 2-3% every 2 weeks as constructs matured. Contrary to our hypothesis, neither decreasing nor increasing load led to further tissue maturation. We hypothesize stepped cyclic load may disrupt cellular tensional homeostasis, leading to repeated remodeling of collagen and shifted proteoglycan accumulation. This study provides insight into how stepped cyclic loading affects hierarchical fiber formation and maturation, which will help to engineer stronger replacements and better rehabilitation protocols.

bioengineering↗

bioRxiv2024

Intermittent Cyclic Stretch of Engineered Ligaments Drives Hierarchical Collagen Fiber Maturation in a Dose- and Organizational-Dependent Manner

AbstractHierarchical collagen fibers are the primary source of strength in tendons and ligaments, however these fibers do not regenerate after injury or with repair, resulting in limited treatment options. We previously developed a culture system that guides ACL fibroblasts to produce native-sized fibers and fascicles by 6 weeks. These constructs are promising ligament replacements, but further maturation is needed. Mechanical cues are critical for development in vivo and in engineered tissues; however, the effect on larger fiber and fascicle formation is largely unknown. Our objective was to investigate whether intermittent cyclic stretch, mimicking rapid muscle activity, drives further maturation in our system to create stronger engineered replacements and to explore whether cyclic loading has differential effects on cells at different degrees of collagen organization to better inform engineered tissue maturation protocols. Constructs were loaded with an established intermittent cyclic loading regime at 5 or 10% strain for up to 6 weeks and compared to static controls. Cyclic loading drove cells to increase hierarchical collagen organization, collagen crimp, and tissue mechanics, ultimately producing constructs that matched or exceeded immature ACL properties. Further, the effect of loading on cells varied depending on degree of organization. Specifically, 10% load drove early improvements in mechanics and composition, while 5% load was more beneficial later in culture, suggesting a cellular threshold response and a shift in mechanotransduction. This study provides new insight into how cyclic loading affects cell-driven hierarchical fiber formation and maturation, which will help to develop better rehabilitation protocols and engineer stronger replacements.

bioengineering↗

bioRxiv2023

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 novel 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 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 new insight into how mechanical cues differentially affect enthesis development, while producing some of the most organized engineered enthesis to date.

bioengineering↗

bioRxiv2022

Temporal Application of Lysyl Oxidase during Hierarchical Collagen Fiber Formation Differentially Effects Mechanics in Engineered Tissues

The primary source of strength in musculoskeletal 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 alignment and fiber diameters by 6 weeks. However, tissue mechanics plateau at 1 MPa, suggesting crosslinking is 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 its not well understood how they form. The objective of this study was to evaluate the effect of exogenous LOX treatment at different stages of hierarchical fiber formation in our culture system to produce functional engineered replacements and to better understand factors effecting collagen crosslink maturation. We found LOXL2 treatment 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 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 fiber organization, increased PYD accumulation, and increased mechanics, ultimately matching the tensile modulus of immature bovine menisci. Statement of SignificanceCollagen fibers are the primarily 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.

bioengineering↗

bioRxiv2021

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

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.

bioengineering↗

bioRxiv2020

An Inducible Model for Unraveling the Effects of Advanced Glycation End-Products in Collagen with Age and Diabetes

PurposeIn 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 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 MethodsUsing 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 mM and 0.75mM) induced with blue light for 40 seconds (riboflavin-465 nm). ResultsWe 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. ConclusionsRiboflavin-465 nm is a promising method to trigger AGE crosslinks 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.

bioengineering↗

bioRxiv2020

Driving Hierarchical Collagen Fiber Formation for Functional Tendon, Ligament, and Meniscus Replacement

Hierarchical collagen fibers are the primary source of strength in musculoskeletal tendons, ligaments, and menisci. It has remained a challenge to develop these large fibers in engineered replacements or in vivo after injury. The objective of this study was to investigate the ability of restrained cell-seeded high density collagen gels to drive hierarchical fiber formation for multiple musculoskeletal tissues. We found boundary conditions applied to high density collagen gels were capable of driving tenocytes, ligament fibroblasts, and meniscal fibrochondrocytes to develop native-sized hierarchical collagen fibers 20-40 {micro}m in diameter. The collagen fibers organize similar to native collagen with native fibril banding patterns and hierarchical fiber bundles 50-350 {micro}m in diameter by 6 weeks. Mirroring fiber organization, tensile properties of restrained samples improved significantly with time, reaching [~]1 MPa. Additionally, tendon, ligament, and meniscal cells produced significantly different sized fibers, different degrees of crimp, and different GAG concentrations, which corresponded with respective native tissue. To our knowledge, these are some of the largest, most organized fibers produced to date in vitro. Further, cells produced tissue specific hierarchical fibers, suggesting this system is a promising tool to better understand cellular regulation of fiber formation to better stimulate it in vivo after injury.

bioengineering↗