Rheology of Heterotypic Collagen Networks - American Chemical

ARTICLE pubs.acs.org/Biomac

Rheology of Heterotypic Collagen Networks Izabela K. Piechocka,†,|| Anne S. G. van Oosten,†,‡,||,§ Roel G. M. Breuls,‡ and Gijsje H. Koenderink*,† † ‡

Biological Soft Matter Group, FOM Institute AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands Department of Physics and Medical Technology, Vrije Universiteit Medical Center, Research Institute MOVE, Amsterdam, The Netherlands

bS Supporting Information ABSTRACT: Collagen fibrils are the main structural element of connective tissues. In many tissues, these fibrils contain two fibrillar collagens (types I and V) in a ratio that changes during tissue development, regeneration, and various diseases. Here we investigate the influence of collagen composition on the structure and rheology of networks of purified collagen I and V, combining fluorescence and atomic force microscopy, turbidimetry, and rheometry. We demonstrate that the network stiffness strongly decreases with increasing collagen V content, even though the network structure does not substantially change. We compare the rheological data with theoretical models for rigid polymers and find that the elasticity is dominated by nonaffine deformations. There is no analytical theory describing this regime, hampering a quantitative interpretation of the influence of collagen V. Our findings are relevant for understanding molecular origins of tissue biomechanics and for guiding rational design of collagenous biomaterials for biomedical applications.

’ INTRODUCTION Collagen is the main constituent of the extracellular matrix (ECM) of animal connective tissues.1 It forms elastic networks of fibrils that confer superior tensile strength. Moreover, the mechanical properties of collagen influence the behavior of cells within tissues.2,3 The biophysical mechanisms underlying the remarkable tensile strength of collagen have been studied extensively using fibrils isolated from tissue or reconstituted from purified collagen. Collagen fibers are rather stiff polymers with a Young’s modulus in the range of 1
800 MPa.4
8 They form viscoelastic networks with elastic shear moduli in the range of 1
200 Pa, dependent on pH, ionic strength, and temperature.9
15 When strained, collagen gels tend to stiffen, which may serve as a mechanism to protect tissues against excessive deformation.10,12,15
18 Previous biophysical research has largely focused on networks of purified collagen I, the most abundant type of collagen in noncartilaginous tissues. However, in vivo, collagen I usually forms heterotypic fibrils together with collagen types II,19,20 III,20
22 or V.23
26 This coassembly is thought to provide a mechanism for regulating fibril diameter. This view is mainly based on studies of collagen I and V, which form heterotypic fibrils whose diameter decreases with increasing collagen V content.23,27
31 In tissues, a similar inverse dependence of fibril diameter on collagen V content is observed. In most adult tissues, collagen fibrils contain only 2
5% collagen V and have a broad distribution of diameters in the range of 40
200 nm.32 In the cornea, however, heterotypic fibrils contain 15
25% collagen V and have a uniform diameter of only 25
34 nm.32
34 Several human connective tissue disorders such as Ehlers-Danlos syndrome30,35
37 are associated with increased collagen V r 2011 American Chemical Society

content, indicating that a correct stoichiometry of collagen I and V is critical for normal tissue function. Moreover, several other pathological conditions such as tumors, atherosclerotic plaques, and scars are also associated with increased levels of collagen V.38 The effect of collagen V on the mechanical behavior of collagenous materials is unknown. The aim of this work is to study this effect in a model system of purified collagen I and V. The formation process of heterotypic fibrils of collagen I and V is relatively well understood. The building block of collagen fibrils is the tropocollagen molecule, a coiled triple helix composed of three R-chains. At 37 °C and under appropriate buffer conditions (neutral pH, appropriate ionic strength, and presence of phosphate), purified tropocollagen spontaneously assembles into fibrils.39,40 Specific, noncovalent interactions lead to a precise axial stagger of each molecule by one-quarter of its length relative to its neighbor, giving the fibers a banding pattern with a periodicity of 67 nm in electron microscopy (EM).41,42 This periodicity remains the same when collagen V is present.25,28,30,43 Several mechanisms have been proposed to account for diameter regulation by collagen V. Collagen is first synthesized as procollagen, which is enzymatically processed to collagen by cleavage of N- and C-terminal propeptides. In contrast with collagen I, collagen V retains a long (∼17 nm) part of its N-propeptide. The collagen V helix is buried inside the fibrils, but the N-propeptide extends outward through the gap zones of the quarter-staggered array. The exposed domains may limit lateral growth of fibrils by Received: April 21, 2011 Revised: June 13, 2011 Published: June 15, 2011 2797

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Biomacromolecules steric or electrostatic hindrance of monomer addition.29,30,43 Yet, the diameter-limiting effect is still partially present when the N-propeptide is removed with pepsin, suggesting an influence of the collagen V helix on diameter regulation.28
31 The longer length of the type V helix compared with the type I helix (321 nm versus 299 nm) could make molecular packing less regular,44 or the larger amount of glycosylated hydroxylysine residues in collagen V may limit lateral growth.31 In this Article, we present rheological measurements on collagen networks reconstituted from purified type I and V collagen, varying the composition and total collagen concentration. Moreover, we examine the microstructure of the networks by fluorescence microscopy and the morphology of the fibrils by atomic force microscopy (AFM) and turbidimetry. We compare the rheological measurements with theoretical models of stiff polymers in an effort to uncover the physical origin of collagen V’s influence on the rheology of hybrid collagen networks.

’ MATERIALS AND METHODS Preparation of Collagen Gels. Rat tail collagen I in 0.02 N acetic acid solution (with telopeptides) was purchased from BD Biosciences (Franklin Lakes, NJ). Lyophilized collagen V from human placenta was obtained from Sigma-Aldrich (St. Louis, MO) and dissolved in 0.02 M acetic acid at 4 °C for 24 h. This collagen V was pepsin-treated and thus lacked telopeptides, consistent with most prior studies of collagen structure and cell physiology in the presence of collagen V. Hybrid collagen networks were prepared from mixed stock solutions having collagen V contents of 0, 10, 20, 50, 80, and 100 wt %. Samples with a final volume of 300 μL and total collagen concentration of 0.5
5 mg/mL were prepared on ice by diluting collagen with 30 μL of minimal essential medium (MEM 10, Gibco BRL, Paisley, Scotland) and water, and adjusting the pH to 7.3 with sodium hydroxide (Sigma-Aldrich) within 4 min to prevent premature polymerization.45 MEM has a nearphysiological ionic strength and pH and contains 1 mM phosphate, thus promoting the formation of fibrils with a native D-banding pattern, as verified by AFM imaging. Samples were polymerized between the plates of the rheometer, which was preheated to 37 °C. For fluorescence microscopy, collagen was mixed on ice in a molar ratio of 7:1 with CNA35,46 a bacterial collagen-binding protein with molecular weight of 35 kDa.47 The CNA35 protein was labeled with Oregon Green and was a kind gift from Dr. M. Merkx (TU Eindhoven, The Netherlands). We checked by confocal microscopy on collagen networks with varying collagen/CNA35 molar ratio that the CNA35 protein did not affect collagen network structure. Moreover, collagen networks with CNA35 looked identical to unlabeled networks in bright field microscopy. The collagen solution was gelled at 37 °C in a glass microchamber, which was sealed to prevent solvent evaporation. For AFM, a 2 mg/mL collagen solution was deposited on freshly cleaved mica coated with nitrocellulose. The sample was polymerized in a moist atmosphere at 37 °C. Because the resulting gel was too dense to allow imaging by AFM, we removed excess material just before imaging by peeling the top of the gel off with filter paper. In this way, only filaments stuck to the mica were exposed, and the filament density was sufficiently low to distinguish individual filaments by AFM. The sample was finally dried with N2. Samples were polymerized for 3 h before analysis. Rheological Measurements. We used a stress-controlled rheometer (Physica MCR 501, Anton Paar, Graz, Austria) with a steel cone (40 mm diameter, 1° angle, 49 μm truncation) and bottom plate. Rheology results were the same in parallel plate geometries of various gap sizes, indicating no-slip boundary conditions. Polymerization was monitored by measuring the increase in the shear moduli with an oscillating shear at a frequency, ω, of 3 rad/s and small strain amplitude,

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γ, of 0.5%. The ratio of the stress response, σ(ω), to the applied strain equals the complex shear modulus, G*(ω) = G0 (ω) + iG00 (ω). The elastic modulus, G0 (ω), represents the in-phase (storage) response, whereas the viscous modulus, G00 (ω), represents the out-of-phase (loss) response. The frequency-dependent moduli of collagen gels were measured by oscillatory tests using γ = 0.5% and ω = 30
0.03 rad/s. The nonlinear moduli were measured by applying a large amplitude oscillatory shear with ω = 3 rad/s and γ increasing logarithmically from 0.5% until sample yielding. To characterize the onset of nonlinearity, we define the critical strain (for strain-stiffening samples) and yield strain (for strain-weakening samples) as the value where σ differs from the product G0γ by >10%, where G0 is the linear elastic modulus.16 Characterization of Network and Fibril Structure. Collagen networks were imaged with a spinning disk confocal microscope (Leica Microsystems, Rijswijk, The Netherlands) using a 488 nm laser (Sapphire 488-30 CHRH, Coherent, Utrecht, The Netherlands) for illumination and a back-illuminated cooled EM-CCD camera (C9100, Hamamatsu Photonics, Herrsching am Ammersee, Germany) for detection. Three-dimensional (3D) stacks were obtained by scanning through the z direction in steps of 0.1 μm over a range of 10 μm with a piezodriven 100/1.3 NA oil immersion objective. Maximum intensity projections were made with ImageJ (http://rsbweb.nih.gov/ij/). We estimated the average mesh size, ξ, for collagen networks with a concentration of 2 mg/mL by measuring the distribution of distances between fibers in binarized images obtained by background subtraction and thresholding of the confocal images.48 Unfortunately, we were unable to perform this analysis for networks below 2 mg/mL collagen (due to network inhomogeneities) or >2 mg/mL collagen (due to insufficient contrast between fibers and background). AFM images were obtained in contact mode with a Dimension V scanning probe microscope (Veeco, Plainview, NY) using a silicon triangle cantilever with 15
30 kHz resonance frequency and 0.01
0.50 N/m nominal spring constant (Veeco). We verified that contact mode imaging did not affect fiber structure by comparing with tapping mode images. The fibril diameter was analyzed in Nanoscope 6.14r1 software (Veeco) from the height, averaging over 20
30 fibrils per experimental condition. We compared the height with the width (determined from Gaussian fits to line profiles across each fibril) and found that the height was typically three to five times larger than the width. This discrepancy is probably partially caused by a profile-broadening effect due to tip
sample convolution. In addition, drying of the fibrils may cause collapse and a reduction in height. In view of these potential artifacts, we measured the mass/length ratio, μ, and fibril radius, r, of the collagen fibrils in their native, hydrated state using turbidimetry. Collagen gels were polymerized in 1 cm path length cuvettes, and the light transmission through the gels over a range of incident wavelengths, λ, between 350 and 650 nm was measured with a UV1 spectrophotometer (Thermo Optek). The transmitted light intensity, It, is lower than the incident intensity, I0, because of light scattering by the turbid collagen network. The optical density, D, of the solution is defined as It/I0 = 10
DL, and the turbidity is defined as τ = D ln(10),49 where L is the path length in centimeters and D is in units of inverse centimeters. For a solution of randomly oriented fibers that are long (>800 nm) and thin (2 mg/mL), the networks are more homogeneous, strain-soften, and the plateau modulus is concentration-independent. A direct comparison of the rheology data with theoretical predictions for stiff polymers suggests that the network elasticity is dominated by nonaffine deformation modes. Unfortunately, there is no tractable analytical expression available that predicts the network modulus in this mechanical regime, hampering a quantitative interpretation of the influence of collagen V. Collagen V may potentially affect 2803

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Biomacromolecules the intermolecular interactions among tropocollagen monomers, which may in turn affect the rheology by changing the rigidity of the fibrils as well the interactions among fibrils. The effect of collagen V on network stiffness is relevant to interpret the role of changed collagen V levels in fetal development and natural tissue regeneration as well as in various connective tissue disorders. Moreover, our findings suggest that collagen composition can be used as a powerful control parameter to design collagen-based biomaterials with controlled mechanical properties.

’ ASSOCIATED CONTENT

bS

Supporting Information. Polymerization of collagen gels monitored by rheology and turbidimetry; frequency dependence of the elastic and viscous shear moduli; quantification of nonfibrillated collagen as a function of total collagen concentration; ratio between the linear elastic modulus of 100/0 and 80/20 collagen gels; number of collagen monomers per cross-section from turbidimetry; calculations of fibril diameter from turbidimetry assuming regular axial and lateral packing; doublelogarithmic plots of elastic and viscous shear moduli versus collagen concentration; and theoretical calculations of elastic shear moduli based on entropic and enthalpic models of stiff polymers; mechanical measurements of single collagen fibrils reported in the literature. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel: +31-20-7547100. Fax: +31-20-7547290. E-mail: g.koenderink@ amolf.nl. Present Addresses §

Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. )

Author Contributions

These authors contributed equally to this work.

’ ACKNOWLEDGMENT We thank S. Duineveld, M. Soares e Silva, M. de Wild, and H. Zeijlemaker (AMOLF) for assistance with experiments; T. Smit and V. Everts (VUMC, Amsterdam), F.C. MacKintosh and C. Broedersz (VU, Amsterdam), C. Storm (Technische Universiteit Eindhoven), and F. Caton (Universit
e de Grenoble, France) for helpful discussions; and M. Merkx (Technische Universiteit Eindhoven) for his gift of CNA35. This work is part of the research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)”, which is financially supported by the “Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)”. ’ REFERENCES (1) Brinckmann, J.; Notbohm, H.; M€uller, P. K. Collagen: Primer in Structure, Processing, and Assembly; Springer: New York, 2005. (2) Bell, E.; Ivarsson, B.; Merrill, C. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 1274–1278. (3) Harris, A.; Stopak, D.; Wild, P. Nature 1981, 290, 249–251. (4) Silver, F.; Ebrahimi, A.; Snowhill, P. Connective Tissue Res. 2002, 43, 569–580.

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