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Article pubs.acs.org/journal/abseba
Collagen Fibril Intermolecular Spacing Changes with 2‑Propanol: A Mechanism for Tissue Stiffness Hannah C. Wells,† Katie H. Sizeland,†,‡ Susyn J.R. Kelly,† Nigel Kirby,‡ Adrian Hawley,‡ Stephen Mudie,‡ and Richard G. Haverkamp*,† †
School of Engineering and Advanced Technology, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand Australian Synchrotron, 800 Blackburn Road, Clayton, Melbourne, Victoria 3168, Australia
‡
S Supporting Information *
ABSTRACT: Materials composed primarily of collagen are important as surgical scaffolds and other medical devices and require flexibility. However, the factors that control the suppleness and flexibility of these materials are not well understood. Acellular dermal matrix materials in aqueous mixtures of 2-propanol were studied. Synchrotron-based small-angle X-ray scattering was used to characterize the collagen structure and structural arrangement. Stiffness was measured by bend tests. Bend modulus increased logarithmically with 2propanol concentration from 0.5 kPa in water to 103 kPa in pure 2-propanol. The intermolecular spacing between tropocollagen molecules decreased from 15.3 to 11.4 Å with increasing 2-propanol concentration while fibril diameter decreased from 57.2 to 37.2 nm. D-spacing initially increased from 63.6 to 64.2 nm at 50% 2-propanol then decreased to 60.3 nm in pure 2-propanol. The decrease in intermolecular spacing and fibril diameter are due to removal of water and the collapse of the hydrogen bond structure between tropocollagen molecules causing closer packing of the molecules within a fibril. We speculate this tighter molecular packing may restrict the sliding of collagen within fibrils, and similar disruption of the extended hydration layer between fibrils may lead to restriction of sliding between fibrils. This mechanism for tissue stiffness may be more general. KEYWORDS: collagen, 2-propanol, isopropanol, scaffold, stiffness, elasticity
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INTRODUCTION Many reconstructive surgical procedures require tissues to be reinforced by the addition of scaffold materials. These materials must be immunologically compatible and capable of being readily incorporated by the body into living tissue. They must also have sufficient strength to perform the task and have appropriate stiffness and elastic properties. These scaffold materials are synthesized from a variety of polymers or biopolymer materials1 or produced by decellularization of native materials. Extracellular matrix materials (ECM) derived from tissues have been successfully used as scaffolds2 including commercially available acellular dermal matrix (ADM) materials produced from various species including porcine, bovine, and human dermal tissue.3 Stiffness is a particularly important property of surgical scaffold materials. There is a need for different stiffness properties but especially for softer more pliable materials. However, for rationally controlling stiffness in collagen materials, a better understanding of the structural and chemical factors that contribute to stiffness is needed. Stiffness of collagen materials is also interesting and important in living animals. Collagen is a key structural component in animals imparting both strength and elasticity to organs.4 Echinoderms, for example, can stiffen in response to an © XXXX American Chemical Society
external stimulus by changing the reversible cross-linking of their interfibrillar matrix.5 Decellularization6 and sterilization treatments used in the processing of native tissue to produce surgical devices may have an impact on the mechanical properties of the materials.7,8 The hydration state affects the stiffness of collagen. Dry collagen fibrils have 3−10-times the Young’s modulus of wet fibrils.9−11 Dehydration with ethanol (at a concentration of 70% or more) causes approximately a 3-fold increase in the Young’s modulus of collagen fibrils.12 Similarly, the elastic modulus dentin, which consists of collagen in a mineral matrix, increases with drying either by ethanol extraction or by air drying.13 Drying type I collagen results in a decrease in Dspacing,14−17 including when the drying is faciliated by pretreatment with ethanol.18 Stiffness may also be affected by cross-linking, and this may be associated with changes in hydration. Cross-linking of tissue by glycation19 or with glutaraldehyde treatment20,21 is known to increase stiffness. Glycosaminoglycans (GAGs), such as chondrotin or dermachondons sulfate, are frequently considReceived: June 27, 2017 Accepted: August 30, 2017 Published: August 30, 2017 A
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Figure 1. Experimental setup for SAXS analysis.
ered cross-linkers and have been assigned a mechanical role.22 Conversely, other GAGs such as hyaluronan do not cross-link and may act to maintain hydration, and therefore, their loss may lead to increased stiffness.23,24 Although cross-linking has been demonstrated as responsible for modifying the stiffness of collagen materials, it follows that the underlying structural arrangement of the fibrils should also influence stiffness. It is known that the strength of collagen tissue materials is due in part to the highly fibrillar nature of type I collagen25 and the tissue’s ability to respond to imposed stresses.26,27 Tear strength of leather and pericardium is correlated with collagen fibril orientation such that, when the collagen fibrils are arranged in parallel or almost-parallel sheets, the material is stronger.28−31 Modeling work has suggested that strain stiffening may occur by increasing fiber alignment.32 Experimental studies have provided some evidence that collagen orientation is an important factor in tissue stiffness.33,34 The structure of the individual collagen fibrils themselves may also influence a tissue’s stiffness because it affects other mechanical properties. Collagen fibril diameter has been correlated with strength in some materials. In human aortic valves, regions of high stress contain larger-diameter fibrils;35 in mouse tendon, fibril diameter increases with loading,36 and in bovine leather, higher strength material has larger diameter fibrils.37 The source material also confers specific mechanical properties to the collagenous scaffolding produced, presumably including stiffness. The age of the animal,30,38 the position on the animal from which the material is taken,39 and the species of animal3,29 all influence a material’s mechanical properties. Here, we investigate stiffness in bovine acellular dermal matrix (ADM) materials when immersed in 2-propanol, the sterilization agent of choice in the industry, and when wet or dehydrated in air.
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soft and pliable. They are then freeze-dried for preservation and storage, and it is in this state that the material is available commercially. Samples of the nominally 4 mm thick material were saturated in a range of 2-propanol/water mixtures (0, 30, 50, 70, 80, 90, and 100% 2-propanol) for at least 1 h before synchrotron-based small-angle X-ray scattering (SAXS) analysis or stiffness testing and maintained in the saturated state during both SAXS and stiffness testing Synchrotron SAXS. Diffraction patterns were recorded on the Australian Synchrotron SAXS/WAXS beamline using a high-intensity undulator source. Energy resolution of 10−4 was obtained from a cryocooled Si(111) double-crystal monochromator, and the beam size (fwhm focused at the sample) was 250 × 80 μm with a total photon flux of approximately 2 × 1012 ph s−1 using a Pilatus 1 M detector with an active area of 170 × 170 mm. Two experimental setups were used. For low q measurements, a sample-to-detector distance of 3371 mm was used with an X-ray energy of 12 keV. For high q measurements, the sample-to-detector distance was 900 mm with an X-ray energy of 15.3 keV. Exposure time for diffraction patterns was 1−5 s, and data were initially processed with Scatterbrain software.40 SAXS analysis was carried out in two directions through the samples. The X-ray beam was passed either through the flat surface of the sample normal to its surface (referred to here as normal) or edgeon to the sample (referred to here as edge-on or cross sections) (Figure 1). For the edge-on measurements, because structure varies through the thickness of a sample, measurements were taken at ∼0.15 mm intervals through the whole thickness of each sample. Fibril Diameter. Fibril diameters were calculated from the SAXS data using the Irena software package41 running within Igor Pro. The data were fitted at the wave vector, Q, in the range of 0.01−0.04 Å−1 and at an azimuthal angle that was 92.5° (over a 5° segment) to the long axis of most of the collagen fibrils. This average direction of the long axis of the collagen fibrils was determined as the position for the maximum scattering intensity of the D-spacing diffraction peaks with azimuthal angle. The “cylinderAR” shape model with an arbitrary aspect ratio of 30 was used for all fittings. There was no attempt to individually optimize this aspect ratio, and the unbranched length of collagen fibrils may in practice exceed an aspect ratio of 30. However, 30 was high enough that using a higher value did not improve the fitting. D-Spacing. The D-spacing was determined from the position of the center of a Gaussian curve fitted to the fifth order diffraction peak taken from the integrated intensity from the azimuthal range from 45° to 135°.
METHODS
Source Material. Commercial SurgiMend (TEI Biosciences/ Integra, New Jersey) ADM was used as the test material. This material is derived from young neonatal (animals less than 5 months old) bovine skins. At this stage, these decellularized materials are very B
DOI: 10.1021/acsbiomaterials.7b00418 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering Orientation Index. The OI is a quantification of the spread of microfibril orientation with 1 indicating parallel microfibrils and 0 indicating randomly oriented microfibrils. OI is defined as (90° − OA)/90°, where OA, the orientation angle, is the minimum azimuthal angle range that contains 50% of the microfibrils42 converted to an index28 using the spread in azimuthal angle of one or more D-spacing diffraction peaks. The peak area was measured, above a fitted baseline, at each azimuthal angle. In many of the diffraction patterns, particularly those measured with the X-ray beam normal to the surface, two peaks were observed in the plot of intensity versus azimuthal angle. In such patterns, the OA calculated using the minimum angle centered on one of these peaks is large and depends on the spacing between the two peaks. It therefore does not accurately reflect the isotropy of the collagen fibrils. Thus, an alternative method to measure the OA was used: the intensities of 5° intervals of azimuthal angle were ranked and a sufficient number of these were summed to give 50% of the total intensity over a 180° range where the total angle covered by the summed intervals becomes the OA. When there is only one peak in the intensity versus azimuthal angle plot, this method gives the same OA as when the OA is calculated by summing the area starting at the center of the peak.28 Another manner of describing this is that the OA for one peak is equivalent to 0.675 of the standard deviation of a Gaussian (if the peak were approximately Gaussian in shape). When there are two peaks, the standard method of finding a combined standard deviation for a single OI value from two Gaussians would not give a good measure of anisotropy because the method depends on the separation of the two Gaussians. A more useful method entails combining the two Gaussians after shifting them so that they are superimposed. This method is not reliant on Gaussian distributions and is, effectively, the method used here. Intermolecular Packing. The intermolecular packing was determined from the SAXS peaks in the q range 0.2−0.7 Å−1, which were fitted using the peak fit function in IgorPro. Stiffness Testing. Stiffness was measured with a three point bend test43 with a TA Texture Analyzer (Stable Micro Systems, Godalming, Surrey, UK) (Figure 2). A jaw width of 48 mm was used with a
concentration, and the bend force was normalized for width (but not thickness). Multiple bending was conducted on some samples.
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RESULTS
The Effect of 2-Propanol/Water Mixtures on Stiffness. In the bending test, one side is stretched and the other side is compressed. Because this is a natural product derived from skin, there are differences in the structure of one side of the material to the other and therefore differences in the bend test curves (Figure 3a). In multiple bending tests (Figure S1), bend stiffness is constant for samples treated with water or low concentrations of 2-propanol but decreases by up to 15% after the first two bends for samples treated with over 70% 2propanol and then decreases more slowly for subsequent bends (Figure S2). The bend modulus is therefore calculated from only the first two bends (one side up, then the other side up). There is a very large increase in bending force (therefore stiffness) as the 2-propanol concentration is increased (Figure 3b), which is more readily compared on a logarithmic scale (Figure 3c). From the bending curves, a “bending modulus”, B, is derived that gives a relative bending stiffness of the material (eq 1)
dF = 4Bb dx
(1)
where dF/dx is the average slope of the bend curve (force/ distance) and b is the width of the sample. The bend modulus for the material soaked in different concentrations of 2-propanol/water increases approximately exponentially with 2-propanol concentration (Figure 4). Small-Angle X-ray Scattering of ADM. The two camera lengths provide SAXS patterns that cover the regions for the structure factor, D-spacing (which is also used for fibril orientation) with a long (3.3 m) camera, and for the intermolecular spacing with a short (0.9 m) camera. Typical diffraction patterns from SAXS analysis with the long camera and the short camera are shown in Figure 5. The Effect of 2-Propanol/Water Mixtures on Collagen Structure. The D-spacing for ADM in different mixtures of 2propanol/water varies only slightly in the range 0−90% 2propanol but then drops sharply for 100% 2-propanol (Figure 6a). At 100% 2-propanol, the D-spacing is similar to that in the dry material (slightly higher, but the dry material is actually just equilibrated to the ambient humidity). The orientation index calculated from the azimuthal spread in intensity of one of the sixth order d-peaks remained within a relatively narrow range, and there is no statistical difference in OI between the values obtained (one-way ANOVA) (Figure 6b). Using the short camera, it was also possible to record the region where the intermolecular spacing between tropocollagen molecules is observed. As 2-propanol is added and the concentration is increased, the peak shifts to a higher q (Figure 7a). Peaks were fitted to these curves to accurately determine the peak positions. The peak position represents the characteristic length and peak area (Figure 7b). Collagen fibril diameter was also calculated from the low q region of the scattering pattern. Fibril diameter decreased with increasing 2-propanol concentration (Table 2). The fibril diameter and intermolecular spacing decreases are closely correlated with the stiffness increase as 2-propanol is added in an approximately logarithmic manner (Figure 8).
Figure 2. Three-point bend test under way.
maximum deflection of 10 mm and a deflection rate of 0.1 mm s−1. For each sample ∼5 mm wide, two measurements were made, the first with the concave (when dry) side facing up and the second with the convex side facing up. With the second test, most samples start by being rather curved because they retain some of the bend of the first test. Three strips of material were measured for each 2-propanol C
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Figure 3. Results of three-point bending tests of ADM with 2-propanol/water mixtures. (a) An example of a bend test (treatment: 70% 2-propanol/ water mixture), where the second bend curve starts above the baseline because the sample is curved upward after turning over at the completion of the first bend. (b) The bend curves on a linear scale for samples in water (0% 2-propanol) up to 100% 2-propanol. (c) The same curves plotted on a logarithmic scale.
“molecules” (which are a triple helix of collagen molecules) are aligned in a collagen fibril and separated from each other by a cylinder of hydration that coats the triple helices. This cylinder forms water-mediated hydrogen bonds with a separation of more than just one water molecule.46,47 The decrease in intermolecular spacing results from the 2-propanol progressively extracting this water, thereby collapsing the hydrogen bond structure between the tropocollagen molecules.47 This leads to a progressive decrease in the distance between the tropocollagen from 15.3 Å in water to 11.4 Å in pure 2-propanol. As the tropocollagen packs closer together, the diameter of the fibril, which is made up of a bundle of these tropocollagens, also decreases. This decrease is from 57.2 to 37.7 nm. If the initial intermolecular spacing is approximately 15 Å in water, then this decrease in fibril diameter would equate to a decrease in intermolecular spacing to around 9.2 Å in pure 2-propanol, which is a little more than that measured for the intermolecular spacing (Figure 9). This difference may be due to the nature of the measurement represented by the fibril diameter analysis. This is calculated as the diameter of a rigid rod, but collagen fibrils are not completely rigid at the scale of this measurement so that the average diameter may appear to be slightly increased if a fibril is not perfectly straight. The intermolecular spacing and fibril diameter that the tissues reached in pure 2-propanol are similar to those in dried material, suggesting that 2-propanol is as effective at removing water from within the collagen fibril as is drying. Such a change has been observed in dehydration of collagen in corneas.48 There is evidence that the effect of 2-propanol drying on the
Figure 4. Bend modulus of ADM with 2-propanol/water mixtures determined from the bend test.
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DISCUSSION The increase in stiffness of ADM with increasing concentrations of 2-propanol is very large and comparable to the stiffness change measured between wet and dry ADM. Dehydrated collagen is well-known to be stiffer than wet collagen.44 The increased stiffness of dry collagen has been attributed to restrictions on the ability of the collagen fibrils to rearrange.45 The trend of increasing stiffness with higher 2-propanol concentrations is mirrored by inverse trends of decreasing intermolecular spacing and fibril diameter, as determined from the SAXS data. The change in fibril diameter is a consequence of the decrease in intermolecular spacing. Tropocollagen D
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Figure 5. Typical SAXS diffraction data for ADM materials: (a) diffraction pattern for a 3.3 m camera, (b) diffraction pattern integrated over all azimuthal angles for a 3.3 m camera, (c) diffraction pattern for a 0.9 m camera, and (d) diffraction pattern integrated over all azimuthal angles for a 0.9 m camera.
Figure 6. (a) D-spacing of collagen in ADM with 2-propanol/water mixtures. (b) Orientation index of collagen in ADM with 2-propanol/water mixtures.
61.48 nm). This further supports the idea that the 2-propanol dries the collagen. Recall that the dry ADM was simply equilibrated to ambient humidity and was therefore not fully dry (water content was not measured). It was not as dry as might be achieved with 2-propanol, so a slightly higher Dspacing for the dry material is to be expected. However, there is a difference between the observed changes in D-spacing and fibril diameter. There is not a progressive decrease in D-spacing with increasing 2-propanol, but rather, an initial slight increase followed by a slight decrease. In contrast, fibril diameter decreases steadily with increasing 2-propanol. It is only at a concentration of over 90% 2-propanol that a large decrease in D-spacing occurs, and this decrease is proportionally much less than the decrease in intermolecular spacing or fibril diameter. The D-spacing is not related directly to the fibril
intermolecular spacing may not be completely reversible. A decrease, albeit small, in the intermolecular spacing of collagen occurred in parchment (animal skins) after treatment with 2propanol, although it was measured after the 2-propanol had been evaporated so presumably was no longer present in the material.49 Other studies of 2-propanol and the structure of collagen in leather have not observed significant changes,50,51 however, the techniques used (FTIR, circular dichroism, melting temperature) are not necessarily sensitive to the level of structural detail available with SAXS,51 and the authors refer to the triple helix remaining intact rather than suggesting any structural changes at the fibrillar level. The D-spacing decreases substantially when pure 2-propanol is used (from 63.6 to 60.3 nm), and this decrease is similar to that observed on drying the material (dried material D-spacing: E
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Figure 7. (a) Scattering pattern integrated over all azimuthal angles in the region of the intermolecular spacing feature with different 2-propanol/ water mixtures. (b) Intermolecular spacing diffraction peak position and area for different 2-propanol/water mixtures.
Table 2. Properties and Structure of ADM with 2-Propanol/Water Mixtures 2-propanol concn 0% 30% 50% 70% 80% 90% 100% dry a
OI (SD) 0.304 0.250 0.320 0.312 0.246 0.289 0.324 0.395
(±0.140) (±0.087) (±0.890) (±0.087) (±0.043) (±0.078) (±0.077) (±0.143)
D-spacing (nm) (SD) 63.63 64.06 64.16 64.05 63.99 63.83 60.30 61.48
mode fibril diameter (nm) (SD)
(±0.43) (±0.60) (±0.56) (±0.38) (±0.33) (±0.34) (±0.27) (±0.56)
57.2 (±5.3) 50.1 (±28.2) 47.8 (±1.1) 48.4 (±3.4) 40.6 (±3.1) 40.6 (±2.4) 37.7 (±13.2) 42.9a (±5.2)
intermolecular spacing (Å) (SD) 15.3 14.4 13.9 13.3 13.0 12.6 11.4 11.6
(±0.01) (±0.01) (±0.02) (±0.01) (±0.05) (±0.01) (±0.01) (±0.01)
bend modulus (kPa) (SD) 0.51 (±0.02) 1.26 (±0.07) 3.17 (±0.22) 6.95 (±0.09) 15.7 (±1.3) 28.2 (±2.4) 102.9 (±6.2) 96.1 (±4.3)
There is some additional uncertainty regarding this value.
Figure 8. (a) Effect of 2-propanol on the stiffness (bend modulus) (blue dashed line), fibril diameter (red solid line) (r2 = 0.91), and intermolecular spacing (black solid line). (b) Bend modulus (log scale) and fibril diameter (r2 = 0.93). (c) Bend modulus (log scale) and intermolecular spacing (r2 = 0.99).
F
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(a) Reprinted wth permission (in modified form) from Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Crystal-structure and molecular-structure of a collagen-like peptide at 1.9-Ångstrom resolution. Science 1994, 266 (5182), 75−81 © AAAS;47 (b) Reprinted with permission from Bella, J., Collagen structure: New tricks from a very old dog. Biochemical Journal 2016, 473 (8), 1001−1025 ©The Author.46
Figure 9. (a) View of the packing of tropocollagen molecules looking down the helix axis. Reference and symmetry molecules are shown in different colors. The spacing between helix centers is ∼15 Å in the hydrated collagen-like peptide. (b) More detailed view of the water molecules (cyan) between collagen helices.
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diameter, but rather, it is orthogonal to this dimension and is related to both the length of the tropocollagen molecule and amount of overlap between collagen molecules. It has been shown previously that the D-spacing decreases in tendon collagen upon drying52 as it does in dermal collagen.53 We cannot explain why the decrease in D-spacing with 2-propanol occurs only at the very highest concentration of 2-propanol.
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Corresponding Author
*E-mail:
[email protected]. ORCID
Richard G. Haverkamp: 0000-0002-3890-7105 Notes
The authors declare no competing financial interest.
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CONCLUSIONS
ACKNOWLEDGMENTS This research was undertaken on the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia. The Australian Synchrotron assisted with travel funding and accommodation. This work was supported by an unrestricted educational research grant from Integra Lifesciences and a grant from the Massey University Research Fund. Vladimir Russakovsky and Bret Jessee from Integra Lifesciences provided the impetus to study this question.
The dehydration of collagen results in stiffer material. Here, we have found how this dehydration affects the fibril structure as the water is removed by air drying or replaced by 2-propanol. We speculate that other alcohols may have a similar effect. Both of these result in a decrease in intermolecular spacing. Although this is only one possible mechanism for altering the stiffness in collagen materials, the very large changes in stiffness that result suggest that it could be a rather important mechanism. Unambiguously measuring the hydration of collagen materials is not simple by other means, and measuring the intermolecular spacing may provide an effective tool for studying the hydration at the fibrillar level. Although other factors have been identified that influence stiffness in collagen, such as the fibril structural arrangement and cross-linking, it is possible that processing changes (including chemical treatments that induce crosslinking) may also alter the equilibrium hydration state of the collagen, which may be a confounding factor in studies of tissue stiffness. Although it had been expected that fibril orientation index would contribute to stiffness because it is known to be important for strength, it was surprisingly not correlated with strength in this study. The D-spacing changes are not fully explained, and there is clearly more to be discovered there.
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AUTHOR INFORMATION
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00418. Bend curves for ADM and slope of bend force over bend displacement divided by sample width for ADM (PDF) G
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DOI: 10.1021/acsbiomaterials.7b00418 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
