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1Department of Biomedical Engineering, Boston University, Boston; and 2Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts
Submitted 24 August 2004 ; accepted in final form 20 December 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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15 times higher than that of the digested samples. Understanding the structure-function relationship of elastin networks and the effects of elastolytic injury on their mechanical properties can lead to the elucidation of the mechanism of elastin fiber failure and evaluation of possible treatments to enhance repair in diseases involving elastolytic injury. storage modulus; loss modulus; cell culture; smooth muscle cell
Originally, elastin repair was thought to occur through a sequential process involving debridement of damaged fibers followed by synthesis of new elastin. Our studies of elastase-induced damage to the ECM of cell cultures led to the identification of a previously unknown repair mechanism, salvage repair, in which damaged elastin is repaired without debridement (28). Using primary neonatal rat aortic smooth muscle cells (NNRSMC), as well as pulmonary fibroblasts, we showed that these elastogenic cells are capable of restoring biochemical and ultrastructural properties to elastin in the ECM after elastase treatment (22, 28, 29). We also reported that recombinant tropoelastin, the precursor to elastin, is incorporated and cross-linked into existing extracellular elastin (12, 27). However, the mechanical properties of the repaired matrices have also not been assessed.
The goal of this study was to measure the mechanical and failure properties of an engineered elastin network and to determine how these properties change with elastase digestion. In an attempt to characterize the static and dynamic mechanical properties as well as the failure properties of engineered material produced by cultured elastogenic cells, we used the NNRSMC culture system to study the effects of elastin degradation on the mechanical properties of the ECM. Moreover, we developed a new system for measuring mechanical properties of stretched tissue based on a previous design (14), which allowed for the measurement of both static and dynamic mechanical properties as well as failure properties of engineered sheets. A new tissue-stretching system was essential because of the difficulty of working with material as thin as the ECM sheets used in this study. Characterizing the changes in the mechanical properties resulting from the degradation of elastin is critical to understanding the pathology of diseases involving elastolytic injury and will provide a necessary basis for future studies assessing the effectiveness of repair processes.
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METHODS |
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The engineered ECM sheets that were used in this study were derived from NNRSMC cultures, which are highly elastogenic (26). NNRSMCs were isolated from 1- to 3-day-old Sprague-Dawley rats and grown in primary culture with Dulbecco's modified Eagle's medium containing 3.1 g/l sodium bicarbonate, 1% sodium pyruvate, 1% penicillin and streptomycin (DV3.7), and 20% fetal bovine serum (FBS) as described previously (27). After subcultivation into first passage in 25-cm2 tissue culture flasks (2 x 104 cells/cm2), the cells were maintained for 6 wk with 5 ml of DV3.7 containing 10% FBS. The medium was changed twice weekly, and the cell cultures were routinely monitored by phase contrast microscopy. After 6 wk in culture, the cells were killed with 5% sodium azide in Puck's saline [in mM: 140 NaCl, 5.4 KCl, 1.1 KH2PO4, 1.1 Na2HPO4, 6.1 glucose (pH 7.4)] (29) and stored at 4°C. Before use, the cultures were infiltrated with a gelatin solution, which, after the gelatin solidified, allowed the cultures to be stripped from the culture flasks. Previous studies that used this technique have shown that the cultures were removed intact (21).
Measurement of Insoluble Elastin
To quantify the elastin in cultures that had been lifted, rectangular pieces of gelatin containing the ECM sheet were heated at 50°C to melt the gelatin, washed several times, and the cell layer was subjected to hot alkali treatment (0.1 N NaOH at 95°C for 45 min) (16). The hot alkali-insoluble residue (elastin) was hydrolyzed in 6N HCl for 24 h at 110°C. Amino acid analysis was performed (Beckman model 6300 with System Gold software, Palo Alto, CA) to confirm the characteristic amino acid composition of the elastin, consisting of the elastin cross-links desmosine and isodesmosine, and more than 80% nonpolar amino acids (26). The amount of elastin was calculated as the sum of the amino acids (in nmol) multiplied by the average amino acid mass of 85 ng/nmol. The elastin content of two cell layers cultured at different times was 73 (SD 13) µg elastin/cm2 of cell layer (n = 2).
Experimental Setup and Data Acquisition
A new uniaxial tissue-stretching system based on a previous design was developed (14). The system consisted of a computer-controlled lever arm with a built-in, large-scale force transducer (model 300B dual-mode lever system, Aurora Scientific, Ontario, Canada) on one side and a more sensitive force transducer (model 403A, Aurora Scientific) on the other. The large-scale force transducer was needed to record the force data for failure tests of the engineered sheets, because the forces during these tests exceeded those that could be measured with the more sensitive force transducer used to record the dynamic mechanics data and stress-strain curves. A LABView (National Instruments, Austin, TX) program was developed to run the system and record data. The data was low-pass filtered at a cutoff frequency of 10 Hz (901P Filter Bank, Frequency Devices, Haverhill, MA) before being sampled by the data-acquisition card (DAQCard-6062E, National Instruments) and connector block (BNC-2110, National Instruments) at a sampling rate of 30 Hz. The test stand for the system was designed with a 22-ml bath and a glass base that fit on top of a hot plate. The hot plate was needed to solubulize the gelatin that surrounds the samples after they are attached to the stretcher systems' feet so that it could be washed away.
The alignment and accuracy of the new system was tested as described previously (33). Specifically, we measured the dynamic mechanical properties of a steel spring, which should have a constant storage modulus over the frequency range of the measurements and little to no loss modulus. The storage modulus varied only slightly with frequency, and the loss modulus was more than one order of magnitude smaller than the storage modulus. The overall hysteresis of the system was 0.05.
Protocol
The ECM sheets were cut into 5-mm-wide by 15-mm-long strips from the sheet of gelatin that was lifted from the culture flasks. Samples were affixed to small metal plates (5 mm x 5 mm) with cyanoacrylate glue while still encased in the gelatin, leaving the working area of the sample at 5 mm x 5 mm. These small metal plates were attached to the force transducers and length controller via steel wires. Once the sample was attached to the system, the bath was filled with 22 ml of PBS and the whole system was placed on top of the hot plate and heated until the gelatin dissolved (50°C). The PBS containing the solubilized gelatin was then removed, and the sample was rinsed three to four times with room temperature PBS using an eyedropper. The bath was then refilled with 22 ml of fresh PBS also at room temperature.
Initially, three baseline quasi-static stress-strain (SS) curves to 25% strain were collected to precondition the sample (strain rate of 0.75%/s). Here, we define stress, T, as the recorded force divided by an estimated cross-sectional area (21. 1 µm thick x 5 mm wide) and strain, 
, as the displacement divided by the sample's initial length, l0, defined as the shortest length at which a change in force was detected (Eqs. 1 and 2, respectively).
 | (1) |
 | (2) |
The estimated cross-sectional area (21.1 SD 2.7 µm) was calculated as an average thickness of several measurements along the length of a fixed control sample at 0% strain. Although the fixing process may have caused the sample to shrink, this value is still within the range (20–30 µm) reported by Toselli et al. (30). This measurement was followed by a dynamic measurement in which length oscillations of amplitude ±5% strain were superimposed on a static operating strain of 20%. To do this, the force was recorded during a broadband displacement signal, which is the sum of six sine waves with frequencies between 0.11 and 3.45 Hz that were chosen according to Yuan et al. (33). Force and length data were recorded for the dynamic measurements for a minimum of seven cycles of the waveform (2 min). After the dynamic measurement, there was a 5-min equilibration period followed by another baseline SS curve to 25% strain and another baseline dynamic measurement. For control samples, the SS and dynamic mechanics measurements were repeated at 5, 10, 20, and 30 min after the last baseline measurement. For treatment of samples with elastase, porcine pancreatic elastase (PPE; Sigma, St. Louis, MO) was added to the bath at a final concentration of 0.06 IU/ml, and the SS curve and dynamic mechanics measurements were repeated at 2, 5, 10, 20, and 30 min after the addition of the enzyme. All samples were then stretched to failure at the end of the 30-min measurement period. This protocol was implemented on 10 control samples and 10 PPE-digested samples.
Morphology
Stretched and unstretched control tissues were processed for microscopy to visualize the effect of stretching on the elastin fibers in the engineered sheets. Samples were fixed in glutaraldehyde and embedded in Epon, according to a method previously described (26).
Data Analysis
Dynamics and stress-strain curve analysis.
The dynamic moduli of the engineered tissue sheets were calculated as a function of frequency for the time points described in the protocol above. Briefly, the dynamic moduli were calculated as follows: force and length data were converted to stress-strain data (Equations 1 and 2) and then converted into the frequency domain by taking the fast Fourier transform (FFT). The complex modulus of the tissue, E*, which is defined as the complex ratio of stress as a function of frequency, T(
), divided by strain as a function of frequency, (), was then determined (Equation 3).
 | (3) |
and the auto power spectrum of . The spectra were calculated on blocks of measured data using 75% overlap and then averaged. The data were taken for several cycles of the waveform to obtain a sufficient number of blocks in the estimate of the spectra. The coherence function was also calculated to assess the quality of the spectra and E*. The real part of the complex modulus, E*, is the storage modulus (E'), representing the portion of the stress that is in phase with the strain and is thus a measure of the ability of the sample to elastically store energy as a function of frequency. The imaginary part of E* is the loss modulus (E'') and this represents the portion of stress that is in phase with strain rate and is thus a measure of the amount of energy loss the sample undergoes as a function of frequency. These were then plotted as functions of frequency and compared before and after elastase digestion.
The complex modulus, E*, was also fit with the single-compartment model, first proposed by Hantos (11) as follows:
 | (4) |
is the angular frequency with units of radians per second, n is the normalized angular frequency [i.e., n = /(1 rad/s)], a normalization that results in a stress unit for G and H, and 
is the stress relaxation exponent defined by the equation:
 | (5) |
Because R and I are generally small, the model described by Eqs. 4 and 5 is called the "constant phase" model because the phase angle of E*, 
, is independent of frequency. The parameters G and H are used to simplify the comparison of the storage and loss moduli during digestion. We compared G and H as a function of elastase digestion and these parameters were used to determine the degradation of the tissues' dynamic mechanics. To account for the variability in the cross-sectional area that was not measured for each sample, each data set was normalized to the stress value at 5% strain for that sample.
To quantify the changes in the SS relationship while elastin was being degraded, SS curves were fit with a two-parameter exponential curve with the following form:
 | (6) |
is strain as a function of time. Taking the log of both sides, this equation can be written into a form in which simple linear regression can be used:
 | (7) |
Failure curve analysis. Each failure test curve was converted to a stress-strain curve, and several indices from each curve were measured for comparison to determine the effect that elastase digestion had on the failure properties of the engineered sheets. The indices that were recorded included the maximum stress, the stress at the first break, the strain at first break, the failure stress, and the failure strain. The way in which these indices were determined is illustrated in Fig. 1 for a control sample.
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RESULTS |
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Stretched and unstretched tissues were treated with elastin stains and compared (Fig. 2). The stained elastin fibers are initially randomly oriented and wavy at 0% strain (Fig. 2A). However, after fixing tissue at 30% strain (Fig. 2B), the majority of the elastin fibers are oriented in the direction of strain and have been pulled taut.
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Figure 3 shows a representative example of the dynamic moduli vs. frequency, at baseline, and after 30 min of elastase digestion. The data points are the actual data generated, and the solid line represents the constant phase model fit to the data. The storage modulus, E', is nearly 20 times larger than the loss modulus, E'', both before and after elastase digestion. The constant-phase model fits the data well in both conditions and the resulting loss, G, and storage, H, modulus coefficient values are shown on the figure. The E' of the sample before elastase digestion is 74 kPa at the lowest frequency and it increases slightly to 78 kPa at the highest frequency. The E'' is more than one order of magnitude smaller, and its value is 4 kPa at the lowest frequency and 8 kPa at the highest frequency.
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24 kPa at the lowest frequency and it increases even less than in the pretreatment case to only 26 kPa. The E'' has been reduced to 1.8 kPa at the lowest frequency and increases to 2.8 kPa at the highest frequency. Representative stress-strain loops for a sample at baseline and after elastase digestion are shown in Fig. 4. The decrease in the slope of the curve after digestion signifies that the sample is generating less stress than it did at baseline for the same strain. This means that the sample is less stiff, corresponding to the loss of intact elastin in the sample that accompanies digestion with elastase. The decrease in the slope of the curve also follows from the dynamic data, as the slope of the SS curve is essentially the tissue quasi-static elastance parameter that is closely related to the dynamic elastance, which decreased with digestion (Fig. 3).
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Figure 5 displays the average data of the constant phase model fit parameters normalized by the stress of the samples at 5% strain at baseline for 10 control samples and 10 elastase-digested samples over the time course of 30 min. This normalization was done to minimize the dependence of the data on the estimated cross-sectional area derived from a single sample. For clarity, these normalized loss (G) and storage (H) modulus coefficients are referred to as Hn and Gn, respectively, and are defined as follows.
 | (8) |
 | (9) |
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Examination of the Gn data reveals that the elastase-digested samples are significantly reduced from their baseline value at time zero by 5 min after the addition of the enzyme. It is important to note that there is no significant difference between the Gn in the control sample and in the elastase-digested samples at any time point. However, two-way repeated-measures ANOVA showed that by 20 min, the Gn data of the elastase-treated group was significantly reduced from the baseline value (P < 0.001). Once again there was a significant interaction between treatment and time in the Gn data (P = 0.002).
The pooled data for the parameters of the exponential fit to the SS curves were plotted (Fig. 6). We normalized the amplitude parameter to minimize its dependence on the estimated area of the samples. The normalized amplitude parameter, An, is defined as the amplitude parameter, A, divided by the stress at 5% strain for that sample (similar to equations 8 and 9). An decreases from baseline for the elastase-digested samples, and by 10 min after the start of the elastase treatment, it is statistically significantly reduced from the control samples. Two-way repeated-measures ANOVA found a significant interaction between time and treatment (P = 0.004) for the An data. The exponential scaling factor, b, is not normalized and does not appear to change for either the control or the elastase-digested samples. In addition, there is no significant difference between the values of the two groups for b at any time point.
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The parameters taken from the failure test data (see Fig. 1) are shown in Fig. 7. The parameters that were statistically significantly different between the control and elastase-treated samples were the maximum stress achieved, the stress at the first break, and the failure stress. All of these indicate that these matrices, which are abundant in elastin, were considerably more resistant to mechanical failure before elastase-digestion than after, because they had a much larger maximum stress, a much larger stress at the first break, and a larger failure stress. There was no difference in the failure strain or the strain at first break between the control and elastase-digested samples.
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DISCUSSION |
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Brief heating to 50°C should have little effect on elastin crosslinking or chemistry, because aorta elastin isolated using NaOH at 95°C was not different in amino acid composition from elastin isolated using cyanogen bromide in formic acid at room temperature (23, 25). Kononov et al. (14) used heating to 55°C to remove agarose from lung tissue strips and showed that it had no effect on the mechanical properties of the tissue.
The NNRSMC culture system has been used previously by our group and is a very useful system for studying elastolytic injury and repair of elastin (12, 28). An important feature of this system is the ability to alter the type and amount of structural macromolecules in the ECM of the culture (4, 3). Furthermore, cell culture models can be conveniently used to carry out experiments requiring prolonged time periods, such as efforts to repair damaged elastin by treatments with recombinant tropoelastin. These cultures (25 cm2 surface area x 73 (SD 13) µg elastin/cm2) contained 1,825 (SD 325) µg elastin compared with our reported values of 1,828 (SD 222) µg (28) and accounted for "up to 50% of the total protein" (28). In another study, elastin content was 47.2% of protein in 6-wk-old cultures (3). Rat aorta contains 40.6% elastin (25). Native lung typically has somewhat lower values for elastin; for example, rat lung has 13.4% elastin (25).
The current study demonstrated the feasibility of measuring the mechanical and failure properties of strips taken from the ECM of this culture system. ECM sheets were derived from cultures that were grown in the absence of ascorbic acid (vitamin C) that resulted in a network comprised of cross-linked elastin and proteoglycans with trace amounts (<1% of total protein) of fibrillar collagen (18). To date, no one has reported the mechanical properties of the ECM of this system. Quasi-static stress-strain relationships, dynamic mechanical properties, and failure properties of the engineered elastin-rich matrices both with and without 30 min of elastase treatment were measured. Because elastase also digests the proteoglycans in the matrix, the mechanical properties measured in this particular study are a function of both elastin and proteoglycan digestion (4, 5).
Elastin is known to have a fairly linear stress-strain relationship (8, 31), and yet all data taken in this study showed a nonlinear toe at the beginning of the stress-strain curve (see Fig. 4). We believe that this nonlinearity in the stress-strain relationship is due to the effects of the reorganization of the elastin network as macroscopic strain increases. This reorganization comes about because the elastin fibers in the ECM of the NNRSMC cultures have no preferred direction and thus the fibers are randomly oriented and have a wavy configuration at 0% strain. As strain in the sheet increases, those fibers that are not initially oriented in the direction of the strain begin to reorient into this direction, and as they do so the waviness decreases (Fig. 2) and the effective spring constant of the system increases, which produces a nonlinear response in stress. Once the majority of the fibers are aligned with the direction of strain, there is a more linear stress-strain relationship as seen in Fig. 1 just before failure occurs.
To compare the SS data at various time points in the protocol and to elucidate the differences in SS data between control and digested samples, the stress-strain data was fit to a two-parameter exponential curve as defined by Eq. 6. As shown in Fig. 6A, the normalized amplitude parameter, An, was significantly decreased after 10 min of digestion. This normalization was used to remove the effects of the uncertainty in the estimated cross-sectional area on the stress. The exponent, b (Fig. 6B), stayed fairly constant throughout the 30-min period and there was no significant difference between the control and digested samples. The decrease in the amplitude constant, An, indicates that digestion caused a decreased total stiffness of the tissue. One can also conclude from these data that digestion had no effect on the nonlinearity of the curve as represented by b. This supports the idea that the nonlinearity in the SS curves of the matrices is related to network effects, because orientation of the elastin fibers in the digested samples should still be random with respect to the direction of strain and thus the nonlinearity associated with fiber reorientation should persist.
One would expect that a sample that contained a high proportion of elastin would have a much larger storage modulus than loss modulus as this represents the component of the complex modulus that is in phase with the strain. Indeed, the Gn data were almost 20 times smaller than Hn (Fig. 5). During digestion, these parameters decreased to 30% of their baseline value such that by 5 min after the addition of elastase, the digested samples had a significantly lower storage modulus coefficient. The loss modulus coefficient was not significantly different between control and digested samples at any time point. This is likely due to the fact that the loss modulus coefficient is very small for these tissues because they contain a high proportion of elastin. Also, the effect of noise on the loss modulus is nearly 20 times larger than on the storage modulus, thus the large variability could obscure any significance. It is important to note, however, that the digested samples did have a significant decrease in Gn from their baseline values by 20 min after treatment. It is possible that some of the decrease in the storage modulus coefficient with time is due to stress relaxation. The stress relaxation function consistent with the constant phase model has a power law functional form (34). The stress relaxation exponent, , of the power law is 0.06 for this material, which corresponds to a 17% decrease in the storage modulus coefficient over the 30-min measurement period (a decrease to 94% of baseline per decade of time over 3 decades). The measured decrease in the storage modulus coefficient in our data was 70%, which means that a large part of the decrease we measure must be related to the effect of the elastase. However, even the 17% contribution due to stress relaxation is likely an upper bound because there was no change in the control data for the storage modulus coefficient within the same time period.
Several groups previously measured the elastic moduli of elastin in arterial wall through both in vivo (1) and in vitro methods (7). The reported values are in the range of 1–5 MPa, more than one order of magnitude larger than those we report here. Both of these studies used a model that assumed that elastin and collagen fibers were in parallel. The elastic modulus of elastin was then calculated by assuming that the low-strain, linear portion of the stress-strain curves in arteries is only an effect of the elastin, which is likely not the case. In fact, Yuan et al. (34) found that collagen contributed significantly to the stress-strain behavior of lung tissue at low prestress. On the basis of their data the result of not including the effects of collagen fibers in the low-strain, linear region could increase the elastic modulus of elastin by a factor of two. In addition, both of these measurements take place with the smooth muscle cells intact. The active contraction of smooth muscle cells can produce an intrinsic prestress on the ECM, which in turn can further increase the estimated modulus of elastin.
Structure also plays an important role in determining the modulus of a fiber network. If the fiber network is entirely random, we could expect a lower modulus compared with a network whose fibers are aligned in the direction of the strain (as in the vessel wall). This is because some of the strain energy developed in the stress-strain curve must go into reorienting the fibers. A better comparison for our tissue modulus would likely be the modulus of purified elastin, which has been reported to be 670 kPa (10). We calculated the static incremental Young's modulus of our samples from the higher strain, linear region of the failure curves where presumably the elastin fibers are aligned with the strain. These modulus values were between 400 and 500 kPa, which is close to the purified elastin modulus mentioned above. This incremental modulus calculation is done near the failure strain and yet the stress is calculated from the thickness at 0% strain and not the decreased thickness near failure. This could alter the calculated modulus by as much as a factor of two, thus the 400–500 kPa range would be an underestimation and the real modulus is likely much closer to the previously reported values.
The failure stress of the control samples was 15 times larger than that of the digested samples (Fig. 7). This is to be expected based on the decreased stiffness of the samples that were digested with elastase. However, it is interesting to note that the failure strain was not different between the control and digested samples. This finding raises some important questions. For example, where might elastin networks break? If the elastin fibers form a continuous network across the sample then the failure of the network may be due to failure of the elastin itself. The reduced failure stress would be a result of destroyed and/or damaged elastin no longer contributing to the buildup of stress, and the failure strain remaining the same after treatment would then likely be a result of some innate critical strain in the elastin molecule, similar to St. Venant's theory of maximum normal strain (6). On the other hand, if the elastin network is not continuous across the sample then microfibrils and/or proteoglycans can also contribute to the failure properties. Microfibrils and proteoglycans in this instance would serve to transfer longitudinal stresses between fibers through shear. The fact that PPE digests proteoglycans (4, 5) would then also contribute to the reduction in failure stress after elastase treatment. In pulmonary fibroblast cultures, microfibrils are exposed by elastase-induced degradation of elastin and may be relatively resistant to elastase (22). In this instance, the elastin fiber network would be expected to fail first. This would result in a lower failure stress, due to fiber destruction and/or degradation, and a similar failure strain as control samples as a result of the innate critical strain of the elastin molecule.
There are some limitations in the new mechanical testing system. Currently the samples are assumed to have an estimated thickness that does not change with increasing strain. This estimated thickness is considered to be the same for all samples. Although the normalization of the dynamic measurements by the stress at 5% strain was able to reduce the spread of the data to some extent, there is still considerable variability in the data that could be generated by the inconsistency of the content of the samples throughout the thickness. Instrumentation is being developed to accurately measure the thickness of the samples during stretching. In future studies we will be able to calculate the true stress as opposed to an estimated engineering stress. This should decrease the sample-to-sample variability that was seen in this study.
In summary, this study was designed to develop the tools and methods to investigate the mechanical properties of engineered ECM sheets derived from NNRSMC cultures as a model for understanding mechanical failure of elastin in pulmonary emphysema and vessel wall aneurysms. Once the new mechanical system was developed and validated, the static and dynamic mechanical properties, as well as the failure properties, of these elastin-rich ECM sheets were characterized before, during, and after 30 min of elastase digestion. Significant decreases in both static and dynamic stiffness were noted after digestion, and overall failure stress in the samples that were digested by elastase was significantly reduced. This new measurement system will likely have wide-ranging applications in evaluating the functional properties of ECM produced by cells under a variety of controlled treatment conditions.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and after 30 min of elastase-digestion (
) with the storage modulus shown in A and the loss modulus is shown in B. Points represent the actual recorded data, whereas the lines are the fits of the constant phase model given in 
