Introduction
Collagen is the most abundant protein in mammals and represents one-third of the human body's proteins [1, 2] . It is the main constituent of various connective tissues, such as tendons and cartilage. Furthermore, the organic matrix of bone tissue is comprised of approximately 90% of type-I collagen [3] . The hierarchical structure of collagen is depicted in Fig. 1 . Three left-handed a-chains, two a 1 (I) and one a 2 (I), twist in a right-handed fashion around one another to form a triple helix called tropocollagen [4] [5] [6] [7] . High concentrations of glycine (Gly) and proline (Pro) have been described as the source of the triple helix conformation. More specifically, a (Gly-X-Y) n sequence is repeated, where the X amino acid is usually Pro and the Y is hydroxyproline (Hyp). It is widely accepted that the a-chains adopt a 10/3 (10 Gly-X-Y triplet units per 3 turns) helix conformation [4, 6, 7] . However, the 7/2 (7 units per 2 turns) helical conformation has been also proposed [5, 8, 9] . The Abbreviations CCD, charged coupled device; DAC, diamond anvil cell; FC, fluorinert TM ; Gly, glycine; His, histidine; Hyp, hydroxyproline; Lys, lysine; meth/ eth, methanol-ethanol mixture; Pro, proline; PTM, pressure transmitting medium; SAXS, small angle X-ray scattering; TAG, triacylglycerol; d, deformation vibration; m, stretching vibration.
Introduction
former is characterized by a 28. 6 A axial repeat [6] whereas in the latter this value is ca. 20 A [5] . The triple helix structure is stabilized by the formation of strong hydrogen bonds between the N-H hydrogen atom of Gly and the carboxyl oxygen of an X (Pro) residue of another chain in the centre of the triple helix [4, 6, 10] . The tropocollagen triple helices have approximate dimensions of 1.5 9 300 nm, leaving gaps of 20 nm to adjacent helices, and coil further around each other to form 500 nm-thick collagen fibrils and finally fibres with a diameter of 20 lm [11] [12] [13] . The specific arrangement of the collagen molecules results in the observation of cross-striations with a period of approximately 65 nm when observed by an electron microscope [5] . Along with the formation of interchain hydrogen bonds (C=OÁÁÁH-N) that stabilize the triple helix [14] , covalent cross-linking of the side chains of lysine (Lys) and histidine (His) residues formed between different tropocollagen units, further strengthens the collagen structure [4, 10, 15, 16] . The cross-links occur usually near the N-and C-termini (nonhelical regions that contain approximately 20 amino acid residues) of the collagen molecules [4] . It should be noted that mutations that alter the triple helix structure result in various disorders [2] .
Introduction
The behaviour of structural proteins, such as collagen, under hydrostatic pressure, is of great interest because it determines, at the molecular level, the mechanical properties of various tissues which are often subjected to high amounts of stress. High hydrostatic pressure can be generated by containing and compressing a sample in a very small volume filled with a fluid (pressure transmitting medium, PTM) that according to Pascal's law, when it experiences a pressure change, transmits the same change throughout its volume. In this work, we present the results of a Raman spectroscopic study of type-I collagen from bovine Achilles tendon that is subjected to hydrostatic pressure up to 4 GPa, by immersion in two different PTM: Fluorinert TM (FC) 1 : 1 70/77 and alcohol, 4 : 1 methanol-ethanol (met/eth) mixtures, in a diamond anvil cell (DAC). Small angle X-ray scattering (SAXS) is used to assess the structural quality of the collagen sample. The mechanical response of tissues is an important and complicated issue as their mechanical properties are strongly dependent on the orientation, distribution and behaviour of the collagen fibres [3, 13, 15] . Therefore, the investigation of the high pressure response of the collagen fibres will contribute towards understanding of the complex mechanical behaviour of hard and soft tissues, as well as their pressure stability region. Raman spectroscopy, an analytical technique that probes vibrational excitations in matter that are intimately related to its structure and properties, is a powerful tool for the investigation of chemical and conformational changes of biological materials [17] . Particularly in the case of collagen, Frushour and Koenig [11] reported its Raman spectrum in comparison with those of gelatin and elastin. Polarized Raman spectroscopy has been also used for the study of oriented collagen fibres by examining, mainly, the amide-III band [18, 19] . Moreover, the study of biological molecules subjected to high pressure by means of Raman spectroscopy provides valuable information about molecular compressibility, protein-protein and ligand-protein interactions and protein denaturation, all important for many scientific fields, such as deep sea research [20] [21] [22] [23] . High pressure Raman measurements on crystalline biological samples, as for example amino acids, have been used for the study of the pressure response of side chains and hydrogen bonding between them [24] , and phase transitions have also been also identified [25, 26] . Furthermore, pressure application on proteins has proven useful in the study of protein folding after denaturation [27, 28] , where the irreversibility of pressure denaturation has been demonstrated. As an example of tissues, the study of the mechanical response of cortical bone upon compression demonstrated that individual mineral and matrix constituents respond differently to external pressure [3] .
Introduction
This study reveals differences in behaviour between peptide bonds and C-H bonds, located in the periphery of collagen molecules, under the application of hydrostatic pressure. All the pressure induced changes are reversible for pressures up to 4 GPa. Finally, a lipid contamination phase is studied as an 'internal reference' and its behaviour under hydrostatic pressure is compared to that of collagen.
Results

Small angle X-ray scattering of collagen fibres
The structural arrangement of the collagen fibres was investigated by SAXS at ambient conditions. A typical scattering pattern is shown in Fig. 2A . The scattering profiles in the meridional and equatorial directions are shown in Fig. 2B . In the meridional direction, a series of maxima arising from the tropocollagen stacking periodicity along the collagen fibril axis (Fig. 1 ) are evident [29] [30] [31] . By exploiting the momentum transfer (q) of the recorded Bragg peaks, the D-spacing of the collagen fibres is easily deduced using the equation:
Small angle X-ray scattering of collagen fibres
q , where n is the order of the maximum and q = 4psinh/k with 2h being the scattering angle and k the X-ray beam wavelength. A D-spacing of 62.7 nm is found. The prominent peak appearing in the equatorial intensity distribution at q = 1.38 nm À1 is ascribed to the presence of triacylglycerols (TAGs), which are the major constituents of lipids. This peak is absent in the scattering patterns of pure collagen reported elsewhere [32] . The lipid molecules are composed of hydrocarbon chains, which are packed in a hexagonal lattice forming lamellar structures [29, 33, 34] . The faint peak that danielhill@example.org. 2.35 nm À1 in the equatorial distribution might be an indication of hexagonal packing of the lipid molecules [35] .
Raman measurements at ambient pressure
The stability of collagen, when immersed in the two different PTM, was initially examined at atmospheric pressure. Fig. 3A shows the Raman spectra at ambient conditions recorded from the collagen sample immersed in the FC and alcohol (meth/eth) mixtures as well as in air. The contribution of the PTM to the Raman signal is also illustrated in Fig. 3A , where collagen bands that do not overlap with those of the two PTM are labelled. Direct comparison of the collagen spectra recorded in air to that immersed in the two PTM clearly indicates that the Raman bands of interest are not affected, implying that no significant interaction between collagen and these PTM is observed at ambient pressure. Three main bands are marked in Fig. 3A , in agreement with the literature [11, 18, 36] . The lower frequency band, centred at approximately 1450 cm
À1
, corresponds to (out of plane) deformation vibrations in the C-H bonds (denoted as d C-H ) located at the side chains of collagen. These bonds cannot be directly assigned to a specific amino acid [37, 38] . The band centred at approximately 1660 cm À1 , called amide-I, is characteristic for proteins and corresponds to vibration of the peptide bond with approximately 80% stretching of the C=O and 20% stretching of the C-N bonds [39] . Due to the coupling between the C=O and N-H modes, the amide-I band is generally sensitive to the hydrogen-bonding pattern of the polypeptide. Therefore changes in the position of the amide-I band correspond to different secondary structures within a protein such as a-helix or b-sheet [36] .
À1
In the case of collagen, about 75% of the Pro carbonyl and Gly amide groups participate in the interchain hydrogen bonds. 10% of the hydrogen bonds are located in the termini zone where the molecular structure is more disordered [14] . According to theoretical calculations, the amide I band in the 10/3 helix is expected to experience a shift towards lower frequencies (red shift) of approximately 10 cm À1 compared to that of the more common a-helix conformation [40] . Finally, the band centred at approximately 2930 cm À1 corresponds to C-H stretching modes and is denoted as m C-H . A tentative assignment of the observed peaks is listed in Table 1 . From Fig. 3A it is evident that when using FC as PTM, all three spectral bands can be safely resolved and monitored upon pressure upstroke but when alcohol is used as PTM, the C-H deformation and stretching modes are considerably masked by the alcohol modes [41] . It should be pointed out that, in the present work, FC and alcohol Comparison of the Raman spectrum of type-I collagen with the spectrum of a contamination phase detected in some sample spots. The secondary phase has been identified as lipids not fully removed during the extraction procedure. The frequencies of the main bands are also marked. All spectra were subjected to background subtraction.
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were selected as PTM because they are commonly used in high pressure spectroscopic experiments. However, the latter, contrary to FC, is polar and it may interact differently with a material like collagen that is stabilized by hydrogen bonding. Although collagen was the dominant constituent, another minor component was also detected in the sample. Its spectrum is characterized by bands similar to those of collagen, as depicted in Fig. 3B . However, differences in the bands' shape and the presence of an additional peak at 1748 cm À1 are observed; the latter being assigned to C=O stretching vibrations. From the spectral characteristics of the secondary phase we infer that it is an adipose tissue that was, most probably, not fully eliminated during the collagen extraction procedure. Its presence was also verified by SAXS as mentioned above. Adipose tissue consists of lipids, mainly TAGs [42, 43] and is also present within the tendons [44, 45] . The spectrum of the contamination phase shown in Fig. 3B exhibits the same characteristics (number, frequency position and relative intensity of the peaks) with the spectra of adipose tissue published elsewhere [46, 47] . We point out that the small size of the laser spot and some different spectral features between the collagen and the lipid phase (relative intensity of C-H stretching band constituents and presence of the C=O peak at 1748 cm À1 ), allowed the discrimination of the two different phases and the acquisition of spectra from sample spots of pure collagen. The lipid contamination phase was also monitored under high pressure.
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Pressure dependence of the Raman spectra of collagen using FC as PTM
À1
To get more insight on the behaviour of the main Raman bands under pressure, the spectra were fitted using Lorentzian lineshapes as illustrated in Fig. 4 .
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Two peaks (P1 and P2) were sufficient to fit the C-H deformation band (d C-H ). In the case of the amide-I band three peaks were used, whereas four or five peaks were necessary to fit the C-H stretching (m C-H ) band. The number of the peaks used was selected according to the criterion that the addition of a peak is justified by an improvement of at least a 5% in the v 2 value normalized to the degree of freedom (which is the number of data points minus the numbers of variable parameters). The peaks P3 and P4 could be assigned to different protein conformations as for example helix and interhelical interactions, b-sheet or turns respectively [48] . The most intense peaks (P1-P7) were tracked with pressure. A tentative assignment, based on the literature [20, 22, 36, [48] [49] [50] [51] [52] , of the peaks used to fit the spectra of collagen is listed in Table 1 . It should be also noted that the strongest Raman peaks of FC at approximately 720 and 750 cm À1 (marked with asterisks in Fig. 3A ) were also followed with pressure and their smooth response suggests hydrostatic pressure behaviour of the PTM. The pressure response of the C-H stretching, m C-H , and bending, d C-H , vibrational modes of collagen is shown in Fig. 5A ,B respectively. The peaks P5, P6 and P7 exhibit a linear pressure dependence with slopes 10.8 AE 0.6, 6.6 AE 0.3 and 5.5 AE 0.7cm À1 /GPa, respectively, that remain constant in the whole pressure range. The peak P2 is also characterized by a linear dependence on pressure with a slope of 2.3 AE 0.3 cm
À1
/GPa that is much smaller compared to the stretching modes. The peak P1 exhibits a different behaviour than P2, with its frequency remaining almost unaffected up to 2 GPa, suggesting that the corresponding molecular vibrations are insensitive to pressure application. Above 2 GPa, the P1 peak shifts towards higher frequencies (blue shift) with a rate of 2.4 AE 0.8 cm
À1
/GPa. The pressure dependence of the P3 and P4 peaks contributing to the amide- I band of collagen is shown in Fig. 6 . Both P3 and P4 peaks are quasi-linearly, within experimental accuracy, red shifted with pressure slopes À2.5 AE 0.6 and À1.2 AE 0.6 cm À1 /GPa respectively.
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Pressure dependence of the Raman spectra of collagen using alcohol as PTM
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As discussed above, the use of alcohol mixture as PTM masks the C-H related vibrations in collagen and therefore only the amide-I band can be monitored. The pressure dependence of the P3 and P4 peaks that comprise the amide-I band is shown in Fig. 7 and is different compared to the case where FC is used as PTM. More specifically, a considerable red shift for the P3 peak with a pressure slope of À7.5 AE 0.6 cm À1 / GPa is found. The P4 peak frequency is less affected by pressure having a slope of À4.7 AE 0.6 cm À1 /GPa up to approximately 1.5 GPa. Above this pressure, it remains constant, revealing that the peptide bond vibrations affected by other than helical conformations, as for example interhelical interactions, are subjected to changes only below this pressure value.
À1
Pressure dependence of the Raman spectra of the lipid phase using FC as PTM Peaks similar to those of collagen, along with an additional one danielhill@example.org cm À1 , were used to fit the spectra of the lipid phase. A tentative assignment of those peaks is listed in Table 2 [43, 46, 49, [53] [54] [55] . The L1 and L2 peaks are attributed to C-H bending modes and correspond to the P1 and P2 peaks of collagen. Similarly, the L6-L8 peaks of the lipids correspond to the P5-P7 peaks of collagen. The peaks L3-L4 and L5 are assigned to C=C and C=O vibrations respectively. The pressure response of the C-H stretching, m C-H , and bending, d C-H , vibrational modes of the lipid phase is shown in Fig. 8A ,B respectively. The frequencies of the L6, L7 and L8 peaks and their pressure dependencies are similar to those of the C-H stretching modes of collagen. As in the case of collagen, the slopes remain constant in the whole pressure range. A different behaviour is observed for the C-H bending modes of the lipid phase. More specifically, the pressure dependence of the L1 and L2 peak frequencies (8.4 AE 1.7 and 10.4 AE 3.5 cm À1 /GPa respectively) is close to each other at low pressure (P < 1 GPa), but with larger slopes compared to the collagen C-H bending modes. Above 1 GPa, the pressure coefficients of the L1 and L2 peaks exhibit an abrupt drop to 3.6 AE 0.5 and 2.5 AE 0.3 cm À1 /GPa respectively. This striking deviant behaviour of the Raman modes further supports the interpretation that this is a second phase in sample. The pressure dependence of the C=C and C=O stretching modes of the lipid phase is shown in Fig. 9 . The L3 and L4 peaks that correspond to 
Discussion
An earlier study of acid soluble collagen extracted from the skin of a 3-month-old pig, in which collagen is not highly cross-linked, suggested that pressure values higher than 0.32 GPa destabilize the collagen structure [56] . However, our study clearly demonstrates much higher pressure range stability for collagen extracted from bovine Achilles tendon. This excludes the possibility of denaturation or other degradation via hydrostatic pressure application up to 4 GPa, as deduced from the reversibility of the Raman peaks. Proteins are characterized by compressibility, defined as b ¼ À 1 V oV oP where V is the molecule's volume and P the hydrostatic pressure applied, with values that range from 5 to 25 9 10 À2 GPa À1 [21, 57] . Volume reduction due to applied pressure is accommodated by changes in the bond lengths, strong reduction in voids [21, 22] and conformational changes, as for example a-helix-to-coil transitions [58] . Discriminating the contribution of the different types of changes in the compressibility of collagen as a result of applied pressure is difficult to sort out from Raman spectra alone. In general, larger pressure coefficients are observed for vibrations associated with bonds that have a more prominent effect on the molecular volume change [22] . Positive pressure coefficients are associated with shortening in the bond distances, causing in turn an increase in the force constants, whereas the opposite holds for bond elongation. Although studies of the effect of compression on the collagen structure are scarce, X-ray scattering and microscopic observations were extensively applied for the study of the behaviour of collagen and collagen-rich tissues under tension. Several mechanisms that contribute to the elongation of the collagen fibres have been proposed. They include straightening of the collagen kinks, which occur in the gap regions of the collagen fibrils in the nonhelical parts of the tropocollagen molecules, molecular gliding within the fibrils, stretching of the triple helices, change in the D-spacing, better orientation of the fibrils, and coil rotation [31, 32, [59] [60] [61] . These changes are also associated with a decrease in the fibril diameter [32] . However, the extension of collagen fibrils inside tendons was much less than the total tendon extension [62] . Due to the elongated structure of the collagen fibres, bending and shear deformation are also expected to occur [63] . In this study, positive pressure slopes were found for the stretching modes of the C-H bonds that extend outwards from the collagen chains and therefore reflect alterations in the chain environment that occur when fibrils are squeezed closer to one another upon pressure application. The absence of abrupt slope changes of the C-H band components and of some bending modes indicates that the fluorinert PTM behaves hydrostatically throughout the studied pressure range and thus the abrupt slope changes observed for some peaks have to be attributed to pressure-induced structural modifications. In the case of the C-H deformation peaks (P1 and P2), the P1 peak frequency appears to be pressure independent for P < 2 GPa, whereas the pressure slope becomes similar to that of the P2 peak (2.3 cm À1 /GPa, in the whole pressure range). The observed differences could be attributed to the complex structure of the collagen fibres and the twisting of the collagen molecules ( Fig. 1 ) that obviously affects in a different manner the various deformation modes (e.g. wagging, rocking, scissoring, etc.). Moreover, a pitch change from 10/3 to 7/2, fibril sliding in order to reduce the D-gaps, kink compression and bending of the fibrils could also account for the peculiar pressure response of the P1 peak frequency. Contrary to the positive slope of the C-H stretching and deformation modes, the amide-I peaks (P3 and P4) exhibit a red shift upon pressure increase in the whole pressure range studied when both FC and alcohol are used as PTM. The pressure slope values for the FC PTM are À2.5 and À1.2 cm À1 /GPa, whereas for the alcohol PTM are quite larger: À7.5 and À4.5 cm À1 / GPa respectively. We can understand the negative pressure slope values by considering that the amide-I band shifts towards lower wavenumbers when the carboxyl oxygen is strongly H-bonded [64] , causing in turn a softening (strength reduction) of the carboxyl oxygen bond. Generally, the hydrogen bonds are considered to be stabilized at high pressures due to the reduction in the distance between the hydrogen-bonded atoms [65] . Furthermore, it has been reported that the presence of alcohol strengthens the hydrogen bonds as it has been deduced by the increase in the population of the backbone hydrogen bonds in the BBA5 peptide, especially those belonging to the a-helix part of the molecule [66] . This is possibly the reason for the considerably larger absolute values of the pressure coefficients of the amide-I band when the alcohol mixture was employed as PTM. It should be also taken into account that the alcohol molecules are expected to be more easily incorporated in the collagen triple helix upon compression, compared to the larger FC molecules. In case of the alcohol PTM, the P3 peak, assigned to the helical conformation of the collagen molecules, appears more sensitive to pressure application, exhibiting a large red shift (À7.5 cm À1 /GPa) in the whole pressure range. On the other hand, the P4 peak, attributed to interhelical interactions and/or peptide bonds at the terminal regions of tropocollagen, which are more disordered, is less sensitive (À4.7 cm À1 /GPa) for P < 2GPa and becomes pressure independent at higher pressures. The abrupt change in the pressure slope of P4 may be attributed to a structural change from 10/3 to 7/2 conformation or compression of the telopeptide region of the tropocollagen molecule, where the kinks exist. The latter case would result in the reduction in the D-gaps. The negative pressure coefficient values found for amide-I here, compare well with those reported in the literature for the a-and b-conformations, whereas a positive coefficient (4 cm À1 /GPa) was found for the unordered structure [22] .
Discussion
Concerning the Raman spectra of lipids that remained in the sample after the extraction procedure, the C-H stretching modes exhibit similar frequency shifts as those of collagen with positive pressure slopes. The C=C stretching modes of lipids, which are observed in the same frequency range as the amide-I band of collagen, exhibit a clear blue shift with a pressure slope of approximately 4.5 cm À1 /GPa in the whole pressure range (Fig. 9) , indicating hardening of the corresponding bonds which are located in the main chains of the lipid molecules. On the other hand, the stretching mode of the C=O bonds, located in the polar heads of the lipid molecules, exhibits an abrupt danielhill@example.org GPa, where its slope changes from negative (À3.9 cm À1 /GPa) to positive (4.3 cm À1 /GPa), becoming similar to that of the C=C bonds. This transition can be attributed to a change in the packing of the lipid molecules from the hexagonal a-form to an orthorhombic (b΄) or triclinic (b) phase [67] . It is known that the triacylglycerol polymorphs follow the Ostwald step rule, where the less stable polymorph (a-form) crystallizes much faster than the more stable b΄-and b-form when crystallization is promoted by supercooling or supersaturation [35] . Pressure application is expected to have similar effect. A change in the pressure slope of the C-H deformation modes ( In conclusion, this work verifies that the Raman study of biomolecules under high pressure provides valuable information on the compressibility of the bonds, variations that take place at the intermolecular level under hydrostatic pressure and the reversibility of such changes upon pressure release. It can be also applied to various molecules of interest in biology, dentistry, dermatology, food industry as well as in transplantation materials and deep sea research. The deformation of collagen upon pressure application appears to be a reversible process up to 4 GPa. This result reveals the rigidity of collagen up to at least this pressure value, which is an important parameter for the response of collagen-rich tissues. Hydrostatic pressure, being an important mechanical stimulus for cartilage, is used as an agent for increasing the metabolic activity of chondrocytes in tissue engineering studies [68] . However, some of the Raman peaks do not exhibit a linear dependence on pressure in the whole pressure range investigated, suggesting the presence of at least two different deformation mechanisms. More specifically, at pressures lower than 2 GPa, there are two main mechanisms for volume decrease: closer packing of fibrils perpendicular to the molecule's axis and D-gap elimination through fibril sliding parallel to molecule's axis and/or compression of the kinks located at the telopeptide, non triple -helical, part of the tropocollagen molecules. These changes coexist with bond compression that prevails at pressures higher than 2 GPa. No change in the pressure slopes of the C-H stretching modes, located at the periphery of the collagen chains is observed. Up to 4 GPa, the amide-I band experiences a red shift when FC is used as PTM. When meth/eth is used as PTM, reinforcement of the interchain hydrogen bonding causes a more pronounced red shift of the amide band. A second phase, coexisting with collagen, has been identified as lipids that were not fully removed during the extraction procedure. The identification of lipids is based on the appearance of the characteristic C=O stretching mode danielhill@example.org. 1750 cm À1 and the relative intensity of the peaks that comprise the C-H stretching band at approximately 2930 cm À1 . Since the behaviour of lipids under high pressure is of interest in food processing industry [69] [70] [71] , the pressure dependence of the Raman peaks of the lipid phase is also studied. It is different from that of collagen and pressure slope changes at approximately 1 GPa are observed.
Materials and methods
The studied sample is type-I collagen from bovine Achilles tendon, in the form of dry white fibres, purchased from Sigma-Aldrich where it was extracted following the procedure described in the work of Einbinder and Shubert [72] . The sample was stored in the refrigerator (4°C) in a dark sealed container and no further treatment was performed. The SAXS patterns were acquired at the I911-4 SAXS beamline of the MAX-II ring in the MAX IV synchrotron radiation facility in Lund, Sweden, using a PILATUS 2 9 5 array detector [73] . The measurements were performed using a wavelength of 0.91 A and the sample-to-detector distance (ca. 2.3 m) was determined using a silver bethenate reference. The assessed q-range is 0.1 to 2.8 nm À1 . The meridional and equatorial intensity distributions were extracted using the SCATTER software [74] after averaging sectors of 35°and 20°respectively. Raman spectra were recorded in the backscattering geometry using a triple monochromator (DILOR XY), coupled with a LN 2 -cooled charged coupled device (CCD), with a spectral resolution of~2.5 cm À1 at the k = 514.5 nm line of an Ar + laser used for excitation. The laser beam was focused to a circular spot of ca. 2 lm diameter by a 50 9 long-working-distance objective, and the laser power, measured before the objective, was 2 mW. The application of high pressure was performed using two pressure transmitting media (PTM), 1 : 1 FC70/77 Fluorinert TM (FC) and 4 : 1 methanol-ethanol (meth/eth) mixtures in a MaoBell type diamond anvil cell (DAC). Pressure increase was achieved mechanically by tightening screws that bring closer the two diamond anvils, reducing the volume in the sample chamber (a hole drilled in a stainless steel gasket, containing the sample and filled with the PTM) and transferring hydrostatically the pressure to the sample by means of the PTM [20] . Pressure was measured by monitoring the R 1 fluorescence line of a small ruby chip and the measurements were conducted up to 4 GPa where the collagen Raman bands were still well resolved. Raman spectra were calibrated using a Neon lamp and fitted by Lorentzian lineshapes after background subtraction.

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