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Introduction: Dissection or rupture of the aorta is accompanied by high mortality rates, and there is a pressing need for better prediction of these events for improved patient management and clinical outcomes. Biomechanically, these events represent a situation wherein the locally acting wall stress exceed the local tissue strength. Based on recent reports for polymers, we hypothesized that aortic tissue failure strength and stiffness are directly associated with tissue mass density. The objective of this work was to test this novel hypothesis for porcine thoracic aorta. Methods: Three tissue specimens from freshly harvested porcine thoracic aorta were treated with either collagenase or elastase to selectively degrade structural proteins in the tissue, or with phosphate buffer saline (control). The tissue mass and volume of each specimen were measured before and after treatment to allow for density calculation, then mechanically tested to failure under uniaxial extension. Results: Protease treatments resulted in statistically significant tissue density reduction (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.003), which in turn was significantly and directly correlated with both ultimate tensile strength (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.03) and tangent modulus (sham vs. collagenase p = 0.007 and sham vs elastase p = 0.03). Conclusions: This work demonstrates for the first time that tissue stiffness and tensile strength are directly correlated with tissue density in proteolytically-treated aorta. These findings constitute an important step towards understanding aortic tissue failure mechanisms and could potentially be leveraged for non-invasive aortic strength assessment through density measurements, which could have implications to clinical care.
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A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome
Traditional tissue dissociation methods for bulk- and single-cell sequencing use various protease and/or collagenase combinations at temperatures ranging from 28-37oC, which cause transcriptional cell-stress that may alter data interpretation. Such artifacts can be reduced by dissociating cells in cold-active proteases, but few studies have shown that this improves cell-type specific transcription, particularly in tissues hyper-sensitive to mechanical integrity and extracellular matrix (ECM) interactions. To address this, we have dissociated zebrafish tendons and ligaments in subtilisin A at 4oC and compared the results with 37oC collagenase dissociation using bulk RNA sequencing. We find that high-temperature collagenase dissociation causes general cell-stress in tendon fibroblasts (tenocytes) similar to results reported in previous studies with other cell types, but also that high temperature specifically downregulates hallmark genes involved in tenocyte specification and ECM production in vivo. Our results suggest that cold-protease dissociation reduces transcriptional artifacts and increases the robustness of RNA-sequencing datasets such that they better reflect native in vivo tissue microenvironments.
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- Award ID(s):
- 2028424
- PAR ID:
- 10652257
- Publisher / Repository:
- Bio-Protocol
- Date Published:
- Journal Name:
- BIO-PROTOCOL
- Volume:
- 15
- Issue:
- 1370
- ISSN:
- 2331-8325
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.21769/BioProtoc.5293
