Collagen matrix stiffness influences fibroblast contraction force (original) (raw)
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Boundary Stiffness Regulates Fibroblast Behavior in Collagen Gels
Annals of Biomedical Engineering, 2009
Recent studies have illustrated the profound dependence of cellular behavior on the stiffness of 2D culture substrates. The goal of this study was to develop a method to alter the stiffness cells experience in a standard 3D collagen gel model without affecting the physiochemical properties of the extracellular matrix. A device was developed utilizing compliant anchors (0.048-0.64 N m −1) to tune the boundary stiffness of suspended collagen gels in between the commonly utilized free and fixed conditions (zero and infinite stiffness boundary stiffness). We demonstrate the principle of operation with finite element analyses and a wide range of experimental studies. In all cases, boundary stiffness has a strong influence on cell behavior, most notably eliciting higher basal tension and activated force (in response to KCl) and more pronounced remodeling of the collagen matrix at higher boundary stiffness levels. Measured equibiaxial forces for gels seeded with 3 million human foreskin fibroblasts range from 0.05 to 1 mN increasing monotonically with boundary stiffness. Estimated force per cell ranges from 17 to 100 nN utilizing representative volume element analysis. This device provides a valuable tool to independently study the effect of the mechanical environment of the cell in a 3D collagen matrix.
Micromechanics of Fibroblast Contraction of a Collagen–GAG Matrix
Experimental Cell Research, 2001
The contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen-GAG matrices over time. We have identified the microscopic deformations developed by individual fibroblasts which lead to the observed macroscopic matrix contraction. Observation of live cells attached to the matrix revealed that matrix deformation occurred as a result of cell elongation. The time dependence of the increase in average fibroblast aspect ratio over time corresponded with macroscopic matrix contraction, further linking cell elongation and matrix contraction. The time dependence of average fibroblast aspect ratio and macroscopic matrix contraction was found to be the result of the stochastic nature of cell elongation initiation and of the time required for cells to reach a final morphology (2-4 h). The proposed micromechanics associated with observed buckling or bending of individual struts of the matrix by cells may, in part, explain the observation of a force plateau during macroscopic contraction. These findings indicate that the macroscopic matrix contraction measured immediately following cell attachment is related to the extracellular force necessary to support cell elongation, and that macroscopic time dependence is not directly related to microscopic deformation events.
Increased stiffness of collagen fibrils following cyclic tensile loading
Journal of the mechanical behavior of biomedical materials, 2018
Alterations in mechanical loading can induce growth and remodeling in soft connective tissues. Numerous studies have measured changes in the collagen structure and mechanical properties of cellularized native and engineered tissues in response to cyclic mechanical loading. However, a recent experimental study demonstrated that cyclic loading also caused significant stiffening and strengthening of acellular collagen constructs. In this work, we developed an anisotropic hyperelastic model of the collagen constructs to investigate whether the measured changes in the tissue-level properties can be attributed to changes in the anisotropic collagen structure or mechanical properties of the collagen fibrils. The model parameters describing the elastic properties, damage properties, and morphology of the fibril were fit to the stress-stretch response measured for the constructs subjected to different preconditioning strains and cycles. The results showed that the changes in the collagen ani...
Journal of Biological Chemistry, 1999
To learn more about the regulation of contraction of collagen matrices by fibroblasts, we compared the ability of lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) to stimulate contraction of floating and stressed collagen matrices. In floating collagen matrices, PDGF and LPA stimulated contraction with similar kinetics, but appeared to utilize complementary signaling pathways since contraction obtained by the combination of growth factors exceeded that observed with saturating concentrations of either alone. The PDGF-simulated pathway was selectively inhibited by the protein kinase inhibitor KT5926. In stressed collagen matrices, PDGF and LPA stimulated contraction with different kinetics, with LPA acting rapidly and PDGF acting only after an ϳ1-h lag period. Pertussis toxin, known to block signaling through the G i class of heterotrimeric G-proteins, inhibited LPA-stimulated contraction of floating but not stressed matrices, suggesting that LPA-stimulated contraction depends on receptors coupled to different G-proteins in floating and stressed matrices. On the other hand, the Rho inhibitor C3 exotransferase blocked contraction of both floating and stressed collagen matrices. These results suggest the possibility that distinct signaling mechanisms regulate contraction of floating and stressed collagen matrices.
Collagen type V modulates fibroblast behavior dependent on substrate stiffness
Biochemical and Biophysical Research Communications, 2009
Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (<10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are present.
Remodeling by fibroblasts alters the rate-dependent mechanical properties of collagen
Acta biomaterialia, 2016
The ways that fibroblasts remodel their environment is central to wound healing, development of musculoskeletal tissues, and progression of pathologies such as fibrosis. However, the changes that fibroblasts make to the material around them and the mechanical consequences of these changes have proven difficult to quantify, especially in a realistic, viscoelastic three dimensional culture environments, leaving a critical need for quantitative data. Here, we observed the mechanisms and quantified the mechanical effects of fibroblast remodeling in engineered tissue constructs (ETCs) comprised of reconstituted rat tail (type I) collagen and human fibroblast cells. To study the effects of remodeling on tissue mechanics, stress-relaxation tests were performed on ETCs cultured for 24, 48, and 72 hours. ETCs were treated with deoxycholate and tested again to assess the ECM response. Viscoelastic relaxation spectra were obtained using the generalized Maxwell model. Cells exhibited viscoelast...
On the mechanobiology of collagen growth and remodelling
2021
How organized collagenous structure can arise and grow from a cluster of cells remains one of the most important basic science questions associated with connective tissue research. Despite more than a century of research, there are currently no widely accepted mechanistic models of formation, growth, and remodeling of collagen fibrils. It has been hypothesized in our research group that collagen monomers and enzymes are in a dynamic equilibrium with existing fibrils. Tensile forces on fibrils can shift this equilibrium and change the balance between molecular association and dissociation. Here, we sought to answer this question: Does fibril strain promote the molecular assembly of collagen? To investigate this question, individual collagen fibrils were stretched to 0%, 4%, and 6% strain between two microneedles and exposed to a subthreshold concentration of fluorescently labeled collagen molecules to quantify molecular association onto the stretched fibrils. It was shown that labeled monomers rapidly incorporate onto all tested fibrils and reach a plateau. The time to reach plateau was significantly faster for the stretched fibrils (15.6, 7.0, and 6.0 minutes for fibrils under 0%, 4%, and 6% strain, respectively). Analysis of the fibril intensity and photobleaching data indicated that the association rate was significantly higher for fibrils under 6% strain compared to fibrils under 0% and 4% strain, increasing the association rate by 100%. It was concluded that mechanical stresses and strains could increase fibril growth by decreasing the activation energy required for reaction between monomers and fibrils and also by setting fibrils in a lower state of energy, increasing the association rate of monomers and fibrils. iii ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Jeffrey Ruberti who taught me how to pursue science properly. I learned to develop a hypothesis and try my best to prove it wrong. Thank you for giving me the opportunity to work in your research group even though I was a mechanical engineer and didn't know much about biology when I started. I would like to thank my parents who have always supported and encouraged me to keep learning. I would like to thank my friends and coworkers-JJ, Ramin, Monica, Ebraheim, Alex, Isabel and everyone else-which made my life as a graduate student fun and exciting. I learned so much from you and had lots of adventures. I would like to especially thank Dr. Paten and Dr. Susilo. I always benefited and developed ideas from our lunchtime discussions. I would like to thank the Bioengineering department and all the amazing people who work there, especially Susan Wilcox which who was always helpful and her love for soccer was also pleasing. I would like to thank Dr. Michael Jaeggli and Dr. Timothy Lannin who I had the opportunity to work with as a teaching assistance during which I discovered a passion for teaching. Last, but not least, I would like to thank my committee, Dr. Charles Dimarzio and Dr. Chiara Bellini who I benefitted from their expertise and knowledge. Thank you all.
Annals of Biomedical Engineering, 1993
We have measured the dynamics of extracellular matrix consolidation and strengthening by human dermal fibroblasts in hydrated collagen gels. Constraining matrix consolidation between two porous polyethylene posts held rigidly apart set up the mechanical stress which led to the formation of uniaxially oriented fibroblast-populated collagen matrices with a histology resembling a ligament. We measured the mechanical stiffness and tensile strength of these ligament equivalents (LEs) as a function of age at biweekly intervals up to 12 weeks in culture using a mechanical spectrometer customized for performing experiments under physiologic conditions. The LE load-strain curve changed as a function of LE age, increasing in stiffness and exhibiting less plastic-like behavior. At 12 weeks, LEs had acquired up to 30 times the breaking strength of 1-week-old LEs. Matrix strengthening occurred primarily through the formation of BAPN-sensitive, lysyl oxidase catalyzed crosslinks. Sulfated glycosaminoglycan (GAG) content increased monotonically with LE age, reaching levels that are characteristic of ligaments. Cells in the LEs actively incorporated [3Hlproline and [35S]suIfate into the extracellular matrix. Over the first three weeks, DNA content increased rapidly but thereafter remained constant. This data represent the first documentation of strengthening kinetics for cell-assembled biopolymer gels and the results suggest that this LE tissue may be a valuable model for studying the cellular processes responsible for tissue growth, repair, and remodeling.