Biology subjects

Szczesny, S.

Publications and source records attributed to Szczesny, S..

6 recordsLinked to original sources

bioRxiv2025

Female Anterior Cruciate Ligaments Exhibit a Muted Mechanobiological Response to Mechanical Loading

Female athletes are significantly more likely to tear their anterior cruciate ligament (ACL) compared to their male counterparts. While there are several potential reasons for this, previous data from our lab demonstrated that female ACL explants have an impaired remodeling response to loading, which may prevent the repair of fatigue damage and lead to increased ACL rupture. The objective of this study was to identify the mechanisms driving the impaired remodeling of female ACLs to cyclic loading, including the role of estrogen. ACLs were harvested from male and female New Zealand white rabbits and cyclically loaded in a tensile bioreactor followed by bulk RNA-sequencing. Additional ACL explants treated with or without estradiol were analyzed using RT-qPCR to determine the regulatory effect of estrogen on markers for tissue remodeling and inflammatory cytokines with cyclic loading. We found that female ACLs exhibited significantly fewer differentially expressed genes (DEGs) in response to loading compared to male ACLs. Additionally, multiple mechanotransduction pathways were enriched with loading only in the male ACLs. While a few estrogen-related pathways were enriched in both male and female ACLs with loading, the expression of tissue remodeling markers was not different between estrogen treatment and vehicle control. Together, our findings highlight specific mechanotransduction pathways that may be responsible for the muted biological response of female ACLs to load, which provides a potential explanation for the increased rate of ACL tears in women.

bioengineering↗

bioRxiv2024

Embryo movement is required for limb tendon maturation

Following early cell specification and tenocyte differentiation at the sites of future tendons, very little is known about how tendon maturation into robust load-bearing tissue is regulated. Between embryonic day (E)16 and E18 in the chick, there is a rapid change in mechanical properties which is dependent on normal embryo movement. However, the tissue, cellular and molecular changes that contribute to this transition are not well defined. Here we profiled aspects of late tendon development (collagen fibre alignment, cell organisation and Yap pathway activity), describing changes that coincide with tissue maturation. We compared effects of rigid (constant static loading) and flaccid (no loading) immobilisation to gain insight into developmental steps influenced by mechanical cues. We show that YAP signalling is active and responsive to movement in late tendon. Collagen fibre alignment increased over time and under static loading. Cells organise into end-to-end stacked columns with increased distance between adjacent columns, where collagen fibres are deposited; this organisation was lost following both types of immobilisation. We conclude that specific aspects of tendon maturation requires controlled levels of dynamic muscle-generated stimulation. Such a developmental approach to understanding how tendons are constructed will inform future work to engineer improved tensile load-bearing tissues.

developmental biology↗

bioRxiv2024

Allograft and Autograft Anterior Cruciate Ligament Reconstructions Exhibit a Similar Biological Response to Cyclic Loading

ObjectiveAnterior cruciate ligament (ACL) reconstruction is one of the most commonly performed orthopaedic procedures. While outcomes are similar in the general patient population, the rerupture rate of non-irradiated allografts are 3-4 times higher than autografts in young active individuals. Previous studies suggest that the difference in clinical performance between graft types is due to impaired remodeling in allografts in response to loading. The objective of this study was to compare the remodeling response of autografts and allografts to cyclic loading. Furthermore, given that allografts are a foreign object and that immune cell signaling affects fibroblast mechanobiology, we compared markers of the immune cell composition between graft types. MethodsACL reconstructions were performed on New Zealand white rabbits, harvested 8 weeks post-surgery, and cyclically loaded to 2 MPa in a tensile bioreactor. Expression of markers for anabolic and catabolic tissue remodeling, as well as inflammatory cytokines and immune cells, were quantified using quantitative reverse transcription polymerase chain reaction. ResultsWe found that the expression of markers for tissue remodeling were not different between allografts and autografts. Similarly, we found that the expression of markers for immune cells were not different between allografts and autografts. ConclusionsThese data suggest that the poor clinical outcomes and impaired remodeling of allograft reconstructions compared to autografts is not due to a difference in graft mechanobiology.

bioengineering↗

bioRxiv2023

Structural Determinants of Tendon Function During Development and Their Sensitivity to Mechanical Stimulation

The load-bearing capabilities of tendon are acquired during neonatal stages of development, characterized by an abrupt increase in multiscale mechanical properties. While prior work has identified numerous changes within the collagenous structure during these developmental periods, the primary structural elements that give rise to this abrupt mechanical functionality, and their mechanobiological sensitivity, remains unclear. To address this gap in knowledge, we leveraged a combination of ultrastructural imaging, biochemical/thermodynamic assays, multiscale mechanical testing, and shear lag modeling to probe the dynamic structure-function relationships and establish their sensitivity to mechanical stimulation during tenogenesis. Mechanical testing and modeling suggested that the rapid increase in multiscale mechanics can be explained by a increasing fibril length and intrafibrillar crosslinking. To test this, we inhibited collagen crosslinking during development and observed a drastic reduction in multiscale mechanical capabilities that was explained by a reduction in both fibril modulus and length. Using muscle paralysis to investigate mechanosensitivity, we observed a significantly impaired multiscale mechanical response despite small changes in fibril diameter and fibril area fraction. While there was no change in crosslinking density, there was a decrease in thermal stability with flaccid paralysis, and our shear-lag model suggested that flaccid paralysis produces a reduction in fibril length and intrafibrillar crosslinking. Together, these data suggest that both intrafibrillar crosslink formation and fibril elongation are critical to the formation of load-bearing capabilities in tenogenesis and are sensitive to musculoskeletal activity. These findings provide critical insights into the biological mechanisms that give rise to load-bearing soft tissue.

bioengineering↗

bioRxiv2022

Cyclic Loading Induces Anabolic and Catabolic Gene Expression in ACLs in a Load-Dependent and Sex-Specific Manner

Anterior cruciate ligament (ACL) injuries are historically thought to be a result of a single acute overload or traumatic event. However, recent studies suggest that ACL failure may be a consequence of fatigue damage. Additionally, the remodeling response of ACLs to fatigue loading is unknown. Therefore, the objective of this study was to investigate the remodeling response of ACLs to cyclic loading. Furthermore, given that women have an increased rate of ACL rupture, we investigated whether this remodeling response is sex specific. ACLs were harvested from male and female New Zealand white rabbits and cyclically loaded in a tensile bioreactor mimicking the full range of physiological loading (2, 4, and 8 MPa). Expression of markers for anabolic and catabolic tissue remodeling, as well as inflammatory cytokines, was quantified using RT-qPCR. We found that the expression of markers for tissue remodeling of the ACL is dependent on the magnitude of loading and is sex specific. Male ACLs activated a tissue remodeling response to cyclic loading below 4 MPa loads but turned off remodeling at 8 MPa. These data support the hypothesis that noncontact ACL injury is a consequence of failed tissue remodeling and inadequate repair of microtrauma resulting from fatigue loading. Conversely, female ACLs downregulate genes responsible for tissue remodeling in response to cyclic loading at all magnitudes, which may explain the increased rate of ACL tears in women. Together, these data provide insight into the remodeling response of ACLs in vivo and potentially offer novel approaches for preventing ACL rupture.

bioengineering↗

bioRxiv2021

A novel, open source, low-cost bioreactor for load-controlled cyclic loading of tendon explants

A major risk factor for tendinopathy is tendon overuse (i.e., fatigue loading). Fatigue loading of tendon damages the extracellular matrix and induces tissue degeneration. However, the specific mechanisms linking tendon fatigue damage with tissue degeneration are unclear. While explant models of tendon fatigue loading have been used to address this knowledge gap, they predominantly employ bioreactors that apply cyclic displacements/strains rather than loads/stresses, which are more physiologically relevant. This is because of the technical complexity and cost of building a load-controlled bioreactor, which requires multiple motors, load cells, and computationally intensive feedback loops. Here, we present a novel, low-cost, load-controlled bioreactor that applies cyclic loading to multiple tendon explants by offloading weights from a single motorized stage. Using an optional load cell, we validated that the bioreactor can effectively provide load-controlled fatigue testing of mouse and rat tendon explants while maintaining tissue viability. Furthermore, all the design files, bill of materials, and operating software are available "open source" (https://github.com/Szczesnytendon/Bioreactor) so that anyone can easily manufacture and use the bioreactor for their own research. Therefore, this novel load-controlled bioreactor will enable researchers to study the mechanisms driving fatigue-induced tendon degeneration in a more physiologically relevant and cost-effective manner.

bioengineering↗