Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas

Category: Basic Sciences/Biologics Introduction/Purpose: New approaches to improve tendon repair after injury are an active area of research. Critical properties of tendons are governed by the production and assembly of fibrillar collagens. Cross-linking of fibrillar collagen is a primary factor in...

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Main Authors: Nicolas Shealy BS, James Rex BS, Amy Bradshaw PhD, Christopher Gross MD
Format: Article
Language:English
Published: SAGE Publishing 2019-10-01
Series:Foot & Ankle Orthopaedics
Online Access:https://doi.org/10.1177/2473011419S00380
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spelling doaj-f7033c7e7d7b4159973d2d46bf937eea2020-11-25T02:42:30ZengSAGE PublishingFoot & Ankle Orthopaedics2473-01142019-10-01410.1177/2473011419S00380Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by TransglutaminasNicolas Shealy BSJames Rex BSAmy Bradshaw PhDChristopher Gross MDCategory: Basic Sciences/Biologics Introduction/Purpose: New approaches to improve tendon repair after injury are an active area of research. Critical properties of tendons are governed by the production and assembly of fibrillar collagens. Cross-linking of fibrillar collagen is a primary factor in determining the function and mechanical properties of the collagen fibers comprising Enzymatic cross-linking by lysyl oxidase in the telopeptide domain of collagen I and III is one determinant of collagen fibril assembly and is the best characterized biochemical cross-link. Transglutaminase catalyzes the modification of lysine residues that in turn form an n-e-glutamyl lysine bond between proteins in the extracellular space. We hypothesize that transglutaminase-dependent modification of collagen in tendons is also a principal determinant of tendon strength and function and is dependent upon tension. Methods: 3-D collagen gels were generated from acid solubilized type I collagen with telopeptides (Advanced BioMatrix). Collagen gels were plated and loaded into a MechanoCulture FX apparatus (CellScale). Gels were subjected to a 10% stretch for 24 hrs at 37°C at 2hz (dynamic) or no stretch, static controls. Gels exposed to enzymatic cross-linking were incubated with either 2.4 ng of recombinant Transglutaminase 2 (Axxora) in a 10 mM Ca2+ solution. Inhibition and labeling of transglutaminase substrates was performed by incubation of collagen gels with 0.2 mM aminopentyl biotinamide in DMSO. Soluble collagen was separated from insoluble collagen by centrifugation at 10,000G. Insoluble fractions were boiled in SDS-Laemmli buffer prior to separation by SDS-PAGE. Collagen in soluble and insoluble fractions was evaluated by Coomassie stain whereas transglutaminase modification was detected via western blot using streptavidin conjugated horse radish peroxidase to detect biotinylated proteins. Results: Evaluation of collagen gels subjected to dynamic versus static stretch revealed minor differences in insoluble collagen incorporation in the two conditions. Notably, higher molecular weight cross-linked forms of collagen appeared to be higher in dynamic versus static gels. In the presence of transglutaminase, differences in higher molecular weight cross-linked forms of collagen, beta-bands, were also detected. Finally, incorporation of biotinylated transglutaminase substrate into collagen alpha bands was enriched in dynamic versus static cultures. Hence, preliminary results support a differential role for transglutaminase modification in collagen under cyclic tension versus static conditions. Conclusion: A better understanding of the role of dynamic stretching and differential tension in the regulation of collagen cross- link formation is predicted to contribute to improved strategies to treat injured tendons.https://doi.org/10.1177/2473011419S00380
collection DOAJ
language English
format Article
sources DOAJ
author Nicolas Shealy BS
James Rex BS
Amy Bradshaw PhD
Christopher Gross MD
spellingShingle Nicolas Shealy BS
James Rex BS
Amy Bradshaw PhD
Christopher Gross MD
Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas
Foot & Ankle Orthopaedics
author_facet Nicolas Shealy BS
James Rex BS
Amy Bradshaw PhD
Christopher Gross MD
author_sort Nicolas Shealy BS
title Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas
title_short Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas
title_full Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas
title_fullStr Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas
title_full_unstemmed Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas
title_sort dynamic stretching of fibrillar collagen enhances cross-linking by transglutaminas
publisher SAGE Publishing
series Foot & Ankle Orthopaedics
issn 2473-0114
publishDate 2019-10-01
description Category: Basic Sciences/Biologics Introduction/Purpose: New approaches to improve tendon repair after injury are an active area of research. Critical properties of tendons are governed by the production and assembly of fibrillar collagens. Cross-linking of fibrillar collagen is a primary factor in determining the function and mechanical properties of the collagen fibers comprising Enzymatic cross-linking by lysyl oxidase in the telopeptide domain of collagen I and III is one determinant of collagen fibril assembly and is the best characterized biochemical cross-link. Transglutaminase catalyzes the modification of lysine residues that in turn form an n-e-glutamyl lysine bond between proteins in the extracellular space. We hypothesize that transglutaminase-dependent modification of collagen in tendons is also a principal determinant of tendon strength and function and is dependent upon tension. Methods: 3-D collagen gels were generated from acid solubilized type I collagen with telopeptides (Advanced BioMatrix). Collagen gels were plated and loaded into a MechanoCulture FX apparatus (CellScale). Gels were subjected to a 10% stretch for 24 hrs at 37°C at 2hz (dynamic) or no stretch, static controls. Gels exposed to enzymatic cross-linking were incubated with either 2.4 ng of recombinant Transglutaminase 2 (Axxora) in a 10 mM Ca2+ solution. Inhibition and labeling of transglutaminase substrates was performed by incubation of collagen gels with 0.2 mM aminopentyl biotinamide in DMSO. Soluble collagen was separated from insoluble collagen by centrifugation at 10,000G. Insoluble fractions were boiled in SDS-Laemmli buffer prior to separation by SDS-PAGE. Collagen in soluble and insoluble fractions was evaluated by Coomassie stain whereas transglutaminase modification was detected via western blot using streptavidin conjugated horse radish peroxidase to detect biotinylated proteins. Results: Evaluation of collagen gels subjected to dynamic versus static stretch revealed minor differences in insoluble collagen incorporation in the two conditions. Notably, higher molecular weight cross-linked forms of collagen appeared to be higher in dynamic versus static gels. In the presence of transglutaminase, differences in higher molecular weight cross-linked forms of collagen, beta-bands, were also detected. Finally, incorporation of biotinylated transglutaminase substrate into collagen alpha bands was enriched in dynamic versus static cultures. Hence, preliminary results support a differential role for transglutaminase modification in collagen under cyclic tension versus static conditions. Conclusion: A better understanding of the role of dynamic stretching and differential tension in the regulation of collagen cross- link formation is predicted to contribute to improved strategies to treat injured tendons.
url https://doi.org/10.1177/2473011419S00380
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