Collagen solutions
Two different solutions of collagen (A and B) were prepared and compared in this study. Collagen type I was extracted from rat tail for both solutions. This method was previously described by Erhmann et al.[117] Briefly, it involved extracting tendons from young albino rats and their dissolution in acetic acid. For collagen solution A, tendons were rinsed in deionised water and dissolved in 0.04 M acetic acid, following a method previously developed by our group.[105] After 72 hours at 4ºC, the solution was centrifuged, degassed in vacuum and sterilized. The final pH of this solution was 3.5. Aqueous solutions were analyzed by the Biuretic Protein Method to determine the collagen concentration. Collagen solution B, a modified method of the above involving lyophilization for improved long- term storage and reproducibility of subsequent collagen solutions, involved dissolving tendons in 0.02 M acetic acid for 72 hours at 4ºC followed by blending on ice to form a homogeneous viscous solution. This solution was frozen at -20ºC and lyophilized. The lyophilized sponge was mixed in a blender with 0.02 M acetic acid at a dry weight to solution ratio of 4mg/ml. The resulting solution was centrifuged and degassed in vacuum followed by sterilization. The B solution had a final pH of 3.7.
Sterilization
Both collagen solutions were placed in dialysis bags (Spectra/Por 1, MWCO : 6 – 8,000) and soaked in acetic acid 0.02 M for one hour followed by one hour in chloroform 1% in water. Dialysis was continued in sterile 0.02 M acetic acid for 4 to 5 days. This solution was changed daily.
Neutral solutions
Neutral solutions were prepared by adjusting collagen solutions to a pH of 6.8 by adding NaOH (1% v/v) on ice and were then immediately made into films with the method described below.
Preparation of collagen thin films
Samples were prepared in the form of thin films by solvent evaporation from collagen solutions poured into non-treated Petri dishes (Fisher Scientific) at room temperature. The volume of solvent was adjusted to obtain films with the same mass per area of collagen. The resulting films were peeled off and used in the following chemical and mechanical studies. Films made similarly on glass cover slides (Bellco Glass inc.) were used for cell culture assays.
Infrared spectroscopy
The infrared spectra of films were collected on a Nicolet, Magna 550 Fourier transform spectrophotometer (Thermo Nicolet, Madison, WI, USA) equipped with a deuterated triglycine sulfate (DTGS) detector and a germanium-coated potassium bromide beam splitter. One hundred scans were routinely acquired with a spectral resolution of 4 cm-1. The ATR mode was employed using a Split Pea attachment (Harrick Scientific Corp., Ossining, NY, USA) equipped with a silicon hemispherical 3 mm-diameter internal reflection element (IRE). To obtain more detailed information about the secondary structure of macromolecules, we carried out the decomposition of the overlapping components under the amide I counter bands. The curve fitting was performed with GRAMS (Salem, NH, USA), using a linear baseline and Gaussian peak-fit components. The position of components was defined by second derivative analysis in compliance with literature data and the bandwidth at half maximum was fixed between 25 and 30 cm-1 which is a typical width range for peaks related to the vibration of carbonyl groups.
Mechanical characterization
Films were cut into strips 50 x 5 mm with a custom made cutter. Thickness was measured with a micrometer (Mitutoyo, Japan). Films were 23 μm thick (±15%) for acidic collagen films and were 35 μm thick (±15%) for neutral films. Tensile tests were performed with an Instron 5848 microtester apparatus (Canton, MA, USA) at a cross-head speed of 5 mm/min. The ends of the strips were placed between pneumatic grips at a pressure of 345 kPa (50
psi). A gauge length of 25 mm was used. Samples were conditioned at room temperature and a relative humidity of 20% for 4 days prior to testing. Samples were weakly stressed to 0.15 N one minute before testing to ensure the removal of crimps and folds. Crosshead displacement was measured with an optical extensometer. Data was recorded with Merlin operating software and analysed with Excel (Microsoft, WA, USA). Results are expressed as mean ± standard deviation.
Cell culture
NIH 3T3 fibroblasts were cultivated in DMEM medium supplemented with 10% foetal bovine serum, glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), fungizone (0.25 μg/ml) (all from Sigma Aldrich, Milwaukee, USA). Cells were routinely maintained at 37ºC in 5% CO2 – humidified atmosphere and harvested by trypsinization when a monolayer was reached. Cells were seeded onto specimens with 3.5x103 cells in 50 μl medium. Medium was added after 1 hour of adhesion. Cells seeded onto cover slides appropriately treated for cell culture were used as a control.
Scanning Electron Microscopy (SEM)
Cell morphology after 24h and 1 week was investigated by SEM. Medium was removed, samples were washed twice in 0.15 M cacodylate buffer and fixed for 30 minutes at 4ºC with Karnowsky solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.15 M cacodylate buffer, pH 7.4). Following fixation, samples were treated for 30 minutes with a 2% osmium tetroxide in 0.15M cacodylate buffer solution. Samples were then dehydrated with graded ethanol (from 50% to 100%), soaked for 30 minutes in hexamethyldisilazane, dried, mounted on appropriate stubs with colloidal silver and sputter-coated with gold- palladium. Images were collected using a JSM-35CF (JEOL) scanning electron microscope.
Cytotoxicity
After 24h and 1 week, cell viability was assessed using the MTT colorimetric assay (Sigma Chemical Co., St. Louis, MO). Briefly, MTT, [3-(4,5 dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide], is reduced to purple formazan by mitochondrial dehydrogenase in cells indicating normal cell metabolism. After each time point, 100 μl of MTT (1 mg/ml in PBS) was added to the medium and samples were incubated for another 4h. After this final incubation, medium was removed and purple formazan crystals were solubilized with 100 μl DMSO at room temperature. The optical density (OD) at a wavelength of 550 nm was determined using an ELISA reader (BioRad mod. 450). All tests were performed in triplicate and repeated twice. The cytotoxicity rates were calculated from the OD readings using controls as 100%. Results are expressed as mean ± standard deviation. Statistical significance was determined by the Student’s t-test (p<0.005).
Results
Infrared spectroscopy
The infrared spectra of proteins exhibit several features characteristic of the molecular organization of these molecules. Generally speaking, amino acids are linked together through peptide bonds giving rise to several infrared active vibration modes such as amide A and B (near 3330 and 3080 cm-1, respectively) and amide I, II, and III (near 1650, 1550 and 1250 cm-1, respectively, see Table 3-1).[118] Several studies have demonstrated that the amide I spectral feature can be used to determine the secondary structure of proteins. This fairly broad spectral component, which arises mainly from stretching vibrations of C=O groups in the peptide groups, is composed of several underlying features - each of them characteristic of a particular protein secondary structure. For example, the antiparallel β-sheet structure of proteins is observed at 1624 cm-1, while the random coil conformation gives rise to an infrared absorption at 1654 cm-1.[119] However, the infrared peak assignment of the features underlying the amide I component in the spectrum of collagen is somewhat different due to the particular triple helical structure of this protein. Nevertheless,
several studies have been performed in order to establish the relationship between the collagen secondary structure and the band shape of the amide I infrared feature.
Peak Frequency (cm-1)
Ref. A B A/Neutral B/Neutral
Helices of aggregated collagen-like peptides
121 1694 1696 1693 1693
Denatured collagen 120 1682 1683 1682 1684
Carbonyl groups from collagen lateral chains
123 1672 1674 1673 1675 Collagen helices 17 1657 1657 1657 1657 Denatured collagen 120 1652 1651 1650 1651 H2O bending mode vibration 123 1643 1643 1641 1642 Denatured collagen 122, 123 1628 1628 1630 1630 Denatured collagen 120 1612 1612 NA NA
Table 3-1 : Assignments of the spectral features underlying the Amide I infrared band. Table 3-1 displays the peak assignments, obtained from literature, for each of the spectral features underlying the amide I peak of collagen samples. These data were thereafter used to curvefit the amide I peak of the infrared spectra of collagen samples recorded in the present work. As can be seen in Table 3-1, the infrared component observed near 1660 cm-1 is assigned to the triple helical hydrogen bonded conformation of collagen chains,[120,121,122] while those observed near 1680, 1650, 1628, and 1612 cm-1 are most likely due to denatured collagen.[123] In addition, the spectral component located near 1695 cm-1 is associated with helices of aggregated collagen-like peptides also found in gelatin.[124,123] Finally, the feature observed near 1640 cm-1 can be assigned to the H2O bending mode vibration. The structural properties of collagen samples were of particular interest in the current study. To study this, the ratio of the integrated intensity of the 1660 cm-1 infrared spectral component over that of the peak at 1630 cm-1 (A1660/A1630) can be used as a sensitive probe to monitor the degree of order within the various protein films. Figure 3.1 shows the curve fitted amide I spectral features of the various collagen samples investigated. As can be seen in this figure, eight components of about 25-30 cm-1 of width at half maximum were required to fit the amide I peak in the infrared spectra of both acidic collagen samples while seven were required for the neutralized samples. This is a clear
indication that the preparation methods for the acidic collagen samples lead to a partial denaturisation of the protein. Indeed, the infrared amide I peak of either native collagen or gelatin (denatured collagen) can be fitted using only four components with geometrical characteristics similar to those utilized in the current study.
Figure 3.1 : Curve fitting of the Amide I infrared peak of collagen samples A and B before (a and b) and after neutralization (c and d).
The peak assignments aforementioned along with comparison of Figures 3.1a and 3.1b indicate that both samples A and B present an almost identical conformation in an acidic environment as the curve fitting is very similar. In addition, the most important underlying feature of the overall amide I peak of both acidic collagen samples is clearly the peak assigned to denatured collagen, therefore demonstrating that the acidic environment drove the collagen conformation to a less ordered structure.
Neutralizing both samples A and B with NaOH clearly modified the secondary structure of collagen as shown in Figures 3.1c and 3.1d as an increase of the 1660 cm-1 band was observed at the expense of the 1630 cm-1 band. It is therefore obvious that neutralization of collagen leads to a more ordered structure. Moreover, this ordering effect seems to be almost identical while neutralizing either A or B collagen samples. This reordering effect could probably be due to a partial reformation of the collagen triple helix in the neutral environment.
These visual observations were confirmed in a more quantitative basis in Table 3-2 where the A1660/A1630 ratios are reported for all collagen samples. As shown in this table, neutralization of samples A and B leads to an increase of the A1660/A1630 ratio from 0.80 to 1.61 and 0.73 to 1.60 respectively, therefore indicating a more ordered protein molecular structure. Area of Bands A B A/Neutral B/Neutral A1660 2.18 2.40 1.71 2.07 A1630 2.98 3.00 1.07 1.29 A1660/ A1630 0.73 0.79 1.59 1.60
Table 3-2 : A1660/A1630 ratios measured from the curve fitted Amide I infrared feature of collagen samples A and B before and after neutralization.
Mechanical properties
Tensile strength and elastic modulus (Figs. 3.2a, b) were not significantly different for acidic collagen films but were affected significantly by neutralization. Neutral films were weaker and less rigid. Maximum elongation (Fig. 3.2c) was basically the same for all samples tested.
Figure 3.2 : Tensile properties of collagen films a) ultimate strength b) elastic modulus c) maximum elongation.
Figure 3.3 shows a typical stress-strain curve of the collagen films. Acidic collagen exhibits a typical reverse exponential shape. Neutral films exhibited a distinct two region curve as previously observed for hydrated collagen gels.[125] The stress-strain curves presented an exponential behaviour in the low stress region followed by a linear region until failure.
Strain (%) 0 2 4 6 8 10 St re ss ( M P a ) 0 20 40 60 80 100 A B A/NaOH B/NaOH A/Neutral B/Neutral
Figure 3.3 : Representative Stress-Strain curves of films made of acidic and neutral collagen films.
Qualitative response of fibroblast NIH 3T3 cells to collagen films
To asses the biological performance of collagen films, the response of fibroblast NIH 3T3 cells grown on acidic (pH = 3.5) and neutral (pH = 6.8) films was investigated. After 24h, cells seeded on both types of acidic collagen showed a rounded morphology indicating an inadequate environment for cell growth (data not shown). No significant morphological changes, with respect to controls, were observed with cells in contact with neutral collagen films for all time periods studied. After 24h, cells were well spread and showed numerous filopodia, indicating appropriate cell adhesion to the surfaces (data not shown). A subconfluent monolayer was present after 1 week of culture. (Fig. 3.4a, b)
Figure 3.4 : Scanning electron microscopy (SEM) analyses of NIH 3T3 cells after 1 week on neutral collagen A (a) and neutral collagen B (b).
Cytotoxicity
MTT results are presented in Figure 3.5. Results obtained after 24h showed a significant decrease in viability of cells seeded on acidic collagen, as was expected, due to low pH. After 1 week, both acidic collagens showed complete cytotoxicity. Cells seeded on neutral films were deemed viable. Even though neutral collagen films revealed a lower percentage compared to control, morphologic analyses showed good cell behaviour. Results are statistically significant. 0 10 20 30 40 50 60 70 80 90 100 A B A/Neutral B/Neutral C e ll v ia b ilit y ( % ) 24 h 1 week
Figure 3.5 : MTT assay testing cytotoxicity of different collagens on NIH 3T3 after 24h and 1 week. Control is considered as 100% viability. All results are significant with respect to control and between pure collagen (A and B) and neutral collagen (A and B) (p < 0.005).
Discussion
Infrared spectroscopy is one of the most powerful methods for studying the protein structure and the conformational mobility of polypeptide chains. Of particular interest is the analysis of the band shape of the Amide I spectral feature which is particularly sensitive to the secondary structure of proteins[120,104].
In connection with the objectives of this study, FTIR spectroscopy enabled characterization of the secondary structure of collagen samples from rat tail tendons obtained using two different methods. Despite sensible differences in the preparation procedures, it turned out that both A and B samples exhibit a very similar structure with an important content of denatured collagen. Not surprisingly, neutralization of these samples promoted a conformational change of the protein structure for both collagen samples demonstrated by a clear increase of the helical content.
In these circumstances, one could hypothesize that the collagen conformational change observed upon neutralization should be accompanied by a modification of mechanical properties, in agreement with previous findings showing that collagen type I fibrils are more stable and possess higher tensile properties at higher pH[126]. Unexpectedly, the present results suggest that the partial disordered to helical transition is accompanied with a decrease of collagen film mechanical properties. The different form of the collagen samples investigated previously[126], and those evaluated in the present study, can be taken into account to explain this opposite behaviour. In addition, one should emphasize that the relative intensity of the infrared peak assigned to the water molecule bending mode vibration near 1640 cm-1 is about 1.5 times more important (Figure 3.1) in acidic collagen samples than in neutralized ones. Therefore, it seems that more water is bound within the acidic collagen films. This bound water has been shown to increase the stiffness of the collagen molecule by facilitating hydrogen bond formation between polar telopeptides in adjacent collagen molecules[127]. Although neutral films were significantly less resistant with respect to ultimate strength, their decreased elastic modulus may be beneficial for vascular tissue engineering as this property, and the inherent compliance, is generally much lower for natural arteries.
As expected, cells seeded on acidic collagen showed a rounded morphology typical of apoptotic cells. In fact, pH is a critical factor for cell growth and in some cases could be associated with apoptosis induction[128]. Even if further analyses are needed to confirm a programmed cell death reaction induced by acid surface contact, there is no doubt that acidic collagen cytotoxicity is caused by the low pH. In fact, in the case of acidic collagen, the increased cell mortality, due to prolonged contact with an inadequate surface, is evident after 1 week. Collagen neutralization decreased toxicity. In fact, cells show spindle-shape morphology similar to control. A lower viability percentage is due to slower cell proliferation when in contact with neutralized collagen, and not to an eventual apoptosis induction as in the case of acidic collagen.
Other experiments have been conducted with similar neutralized collagen films supplemented with an adequate amount of cell culture medium (data not shown). These conditions allowed the same proliferation rate as the control. Unfortunately, although this method has been shown to work extremely well for gels, dried films were too inhomogeneous and brittle to consider for chemical and mechanical testing. Consequently, this solution has been discarded for the time being.
Our group has previously shown that B collagen films display good biological results with other cell types as well as good results when in contact with blood[129]. In fact, smooth muscle cells and endothelial cells displayed good cell spreading and proliferation on neutral collagen films. Furthermore, collagen films presented low thrombogenicity and displayed low platelet adhesion during static assays.
Conclusion
Thus, we can conclude that lyophilization and mechanical blending during the synthesis of collagen stock solution has no significant effects on final collagen properties and therefore offers a method for obtaining solutions with homogeneous and modifiable collagen concentrations and with more favourable storage capabilities. Neutralizing stock solution prior to solvent evaporation provided films that had lower mechanical properties but significantly improved biological performances. These findings are very encouraging for future applications of this collagen for biomedical applications, most notably for vascular tissue engineering scaffolds.
The above section presented results validating the collagen extraction and processing method. The following section presents more validation results pertaining to the biological and blood contact performances of the collagen validated in the above section.