**6.2 Diffractographic and Raman scattering technical procedures**

XRD measurements were conducted at the Laboratory of Crystallography, Solid State and Materials, School of Chemistry (DETEMA), with a CuKα radiation source of wavelength λ = 1.5418 Å, using a Rigaku Ultima IV diffraction system. The incident ray is calibrated to arterial vessels in a range for 2θ between 5 ° and 60 °, step scan of 0.1 ° for 10 sec. each. The respective diffraction profiles (FVS vs. CVS) were filed for later analysis. Comparative profiles for amnion (FAS vs. GAS; and FAS vs CAS) were treated the same way as having been calibrated for 2θ between 5º and 60 º ranges scanning with steps of 0.2° for 10 sec each.

Raman spectra were recorded using a Raman DeltaNu Advance 532 spectrometer with a laser frequency doubled Nd: YAG, 100mW, with a 532 nm wavelength, scanning in the 200 and 3400 cm-1 region.

X Ray Diffraction: An Approach to Structural

according to the following formula:

**7.1 Cryopreserved vascular tissues results** 

of 1.

**7. Results** 

CVS). See Figure 5

Quality of Biological Preserved Tissues in Tissue Banks 451

Fig. 4. DPS from two tissues categories A vs B to be studied by X-ray diffraction.

The ratio DPS (+) values vs DPS (-) values define the Ordering Profile Coefficient (OPC)

OPC absolute values are always above 0 and they are greater than 1 when +DPS values > - DPS values. When +DPS values < -DPS, OPC falls into an interval greater than 0 and lower

Analysis of the diffraction curves shows that regardless of the condition FVS or CVS, the same design with a peak of maximum intensity to 31.3 º and another lower, at 42 ° in 2θ is kept, whether there are noticeable differences in design profiles between the two categories, even for a single donor. (Perez Campos, 2008). Comparatives diffraction profiles shows the confirmation of a peak intensity for 2θ = 31.3 ° corresponding a d - spacing = 2.86 Å. The lower peak intensity, obtain d – spacing = 2.15 Å applying Bragg Low calculus. This behavior is independent of the vessel (aorta or carotid) and the processed sample (FVS or

(3)

#### **6.3 Planimetric analysis: Obtaining the order coefficients for XRD (Perez Campos, 2008)**

Given the diffraction profiles of two tissues A and B to compare tests, we can define the respective planimetric surfaces, defined under the corresponding diffraction curve, that are a function of the degree of molecular arrangement of the studied tissue.

Fig. 3. Diffractive profiles from two different tissues categories A vs B.

Relative Differential Intensity values (RDIV) established for each 2 θ point between 5º and 60º, will produce a result that: if absolute value tissue A > tissue B will have a resulting positive (+) value; but if tissue A < tissue B will have a resulting negative (-) value.

With those values obtained in each point from 2θ it can be developed Differential Planimetric Surfaces (DPS) that represent the sum of every relative intensity value.

Its mathematical expression is given by the equation:

$$\Sigma\_{\mathbf{A}}(\mathbf{x}^{\bullet}\mathbf{\hat{1}}\mathbf{\hat{1}}\mathbf{\hat{1}}\mathbf{\hat{1}}\mathbf{\hat{1}}\mathbf{\hat{1}}) \propto \mathbf{I}\_{\mathbf{A}\rightarrow\mathbf{B}}\left[\mathbf{\hat{2}}\,\theta\,\mathbf{\hat{1}}\right] \tag{2}$$

Where ( ) represents ordering diffractometric intensity for *y* axes values of tissue A in one point in 2θ; ( ) represents ordering diffractometric intensity for *y* axes values of tissue B, in the same point in 2 θ; I A - B is the difference between each respective ordering diffractometric intensity for *y* axes value at the same point in 2θ. Finally, [2θ (º)] is each point in **x** axes between 5º and 60º angular incidence.

DPS can be edited in a Cartesian model too, where *x* axis is 2θ values and *y* axes is the Intensity Relative Differential Values (IRDV) between both comparative samples for each point 2θ values. (See figure 4)

Given the diffraction profiles of two tissues A and B to compare tests, we can define the respective planimetric surfaces, defined under the corresponding diffraction curve, that are

**6.3 Planimetric analysis: Obtaining the order coefficients for XRD (Perez Campos,** 

a function of the degree of molecular arrangement of the studied tissue.

Fig. 3. Diffractive profiles from two different tissues categories A vs B.

Its mathematical expression is given by the equation:

point in **x** axes between 5º and 60º angular incidence.

point 2θ values. (See figure 4)

Relative Differential Intensity values (RDIV) established for each 2 θ point between 5º and 60º, will produce a result that: if absolute value tissue A > tissue B will have a resulting

With those values obtained in each point from 2θ it can be developed Differential

Where ( ) represents ordering diffractometric intensity for *y* axes values of tissue A in one point in 2θ; ( ) represents ordering diffractometric intensity for *y* axes values of tissue B, in the same point in 2 θ; I A - B is the difference between each respective ordering diffractometric intensity for *y* axes value at the same point in 2θ. Finally, [2θ (º)] is each

DPS can be edited in a Cartesian model too, where *x* axis is 2θ values and *y* axes is the Intensity Relative Differential Values (IRDV) between both comparative samples for each

Σ ( - ) x I A - B [2 θ (º)] = DPS (2)

positive (+) value; but if tissue A < tissue B will have a resulting negative (-) value.

Planimetric Surfaces (DPS) that represent the sum of every relative intensity value.

**2008)** 

Fig. 4. DPS from two tissues categories A vs B to be studied by X-ray diffraction.

The ratio DPS (+) values vs DPS (-) values define the Ordering Profile Coefficient (OPC) according to the following formula:

$$\text{\u00\%} \quad \frac{\text{\u00\%} - \text{\u01\%}}{\text{\u00\%} \quad \text{\u01\%}} \tag{3}$$

OPC absolute values are always above 0 and they are greater than 1 when +DPS values > - DPS values. When +DPS values < -DPS, OPC falls into an interval greater than 0 and lower of 1.

#### **7. Results**

#### **7.1 Cryopreserved vascular tissues results**

Analysis of the diffraction curves shows that regardless of the condition FVS or CVS, the same design with a peak of maximum intensity to 31.3 º and another lower, at 42 ° in 2θ is kept, whether there are noticeable differences in design profiles between the two categories, even for a single donor. (Perez Campos, 2008). Comparatives diffraction profiles shows the confirmation of a peak intensity for 2θ = 31.3 ° corresponding a d - spacing = 2.86 Å. The lower peak intensity, obtain d – spacing = 2.15 Å applying Bragg Low calculus. This behavior is independent of the vessel (aorta or carotid) and the processed sample (FVS or CVS). See Figure 5

X Ray Diffraction: An Approach to Structural

curve. (See Figure 7)

Intensity RV.

Quality of Biological Preserved Tissues in Tissue Banks 453

in FAS d spacing = 3.24 (28.4 ° in 2θ) and in GAS, d spacing = 3.28 (28 ° in 2θ). A second peak is shown to both kinds of samples FAS and GAS for same d spacing = 2.35 (40.4º in 2θ). Contrary to the notable profiles differences showing in the two categories of vascular tissues (FVS and CVS), both amnion profiles -fresh and glycerolized- have almost the same design

Fig. 7. Diffractive curves FAS and GAS profiles. Note: The 1st and 2nd maximun peak labels of each respective diffractive curve indicate; Categories of tissue: 2 Theta value; and

Mean diffractographic profiles for 6 FAS vs 6 GAS let us obtain DPS picture and calculate

OPC values = 14.76 (See respective Figure: 7 and Table: 1)

Fig. 8. DPS profile by FAS vs GAS analysis obtained.

Fig. 5. Diffractografic profiles from thoracic descending aorta. Code color: FVS, Red; CVS, Blue. Note: The 1st and 2nd maximum peak labels of each respective diffractive curve indicate; Category of tissue: 2 Theta value; and calculated d spacing.

Calculated OPC values in respective analyzed vessels, shows a greater crystalline framework for CVS vs FVS, regardless the kind of arterial segment: aorta or carotid. 75% of aortic samples showed OPC values > 1 and 62,5% of carotid samples had the same behavior. Perez Campos et al (2008). Figure 6 show DPS defined from diffractografic FVS and CVS of Figure 5:

Fig. 6. DPS profile FVS vs CVS in a descending thoracic aorta from a male donor 50 years old.

Note the great difference of design shape of DPS curve related to the same one obtained from amnion tissues; (see below).

#### **7.2 Glycerolized amnion tissues results**

The diffraction curves of the glycerolized amniotic membrane, also shows the same kind of form and design for both the FAS and GAS. Notwithstanding the maximum diffractive peak

Fig. 5. Diffractografic profiles from thoracic descending aorta. Code color: FVS, Red; CVS, Blue. Note: The 1st and 2nd maximum peak labels of each respective diffractive curve

Calculated OPC values in respective analyzed vessels, shows a greater crystalline framework for CVS vs FVS, regardless the kind of arterial segment: aorta or carotid. 75% of aortic samples showed OPC values > 1 and 62,5% of carotid samples had the same behavior. Perez Campos et

Fig. 6. DPS profile FVS vs CVS in a descending thoracic aorta from a male donor 50 years old. Note the great difference of design shape of DPS curve related to the same one obtained

The diffraction curves of the glycerolized amniotic membrane, also shows the same kind of form and design for both the FAS and GAS. Notwithstanding the maximum diffractive peak

from amnion tissues; (see below).

**7.2 Glycerolized amnion tissues results** 

al (2008). Figure 6 show DPS defined from diffractografic FVS and CVS of Figure 5:

indicate; Category of tissue: 2 Theta value; and calculated d spacing.

I N T E N S I T Y A.U in FAS d spacing = 3.24 (28.4 ° in 2θ) and in GAS, d spacing = 3.28 (28 ° in 2θ). A second peak is shown to both kinds of samples FAS and GAS for same d spacing = 2.35 (40.4º in 2θ). Contrary to the notable profiles differences showing in the two categories of vascular tissues (FVS and CVS), both amnion profiles -fresh and glycerolized- have almost the same design curve. (See Figure 7)

Fig. 7. Diffractive curves FAS and GAS profiles. Note: The 1st and 2nd maximun peak labels of each respective diffractive curve indicate; Categories of tissue: 2 Theta value; and Intensity RV.

Mean diffractographic profiles for 6 FAS vs 6 GAS let us obtain DPS picture and calculate OPC values = 14.76 (See respective Figure: 7 and Table: 1)

Fig. 8. DPS profile by FAS vs GAS analysis obtained.

X Ray Diffraction: An Approach to Structural

Fig. 10. DPS planimetric picture FAS vs CAS.

Table 2. OPC values FAS vs GAS

**7.4 Cryopreserved amnion tissues results: Raman Spectra** 

between both categories, the showed range is 1000 – 2000 cm-1 in *x*.

positive Intensity values (AU) between FAS (red line) and CAS (blue line).

DPS pictures.

CAS respective diffractive curves:

Quality of Biological Preserved Tissues in Tissue Banks 455

Figure 10: show DPS profile obtained from operative subtractions analysis between FAS Vs

Operative planimetric values DPS obtained showed an OPC = 42.02 (See Table 2).

Newly highlight the notable differences in shape and form between vascular and amnion

OPC CALCULATION FAS vs GAS

∑ DPS + VALUES 102887.67

∑ DPS - VALUES -2448,33

OPC VALUE = (+DPS) / (-DPS) = 42.02

Figure 11 Show Raman Spectra profile from FAS vs CAS assays. Note that having regard to the best imaging definition, and taking account the meaningful change area of Raman Shift

It should be highlighted that arrows points marked correspond to noticeable differences in

OPERATION ABS. VALUES


Table 1. OPC value from FAS vs GAS.

#### **7.3 Cryopreserved amnion tissues results: X-ray diffraction**

4 FAS vs 2 CAS was analysed. The same phenomena of Glycerolized amnion about form and design maintenance, was observed between FAS and CAS. Equally, there are a maximum peak in FAS d spacing = 3.26 (28.2º in 2θ) and in CAS, the same d spacing. Also a second peak is detectable to FAS in d spacing = 2.36 (40.8º in 2θ) and to CAS with equal values. (See Fig 9)

Fig. 9. Diffractive curves FAS and GAS profiles. Note: The 1st and 2nd maximun peak labels of each respective diffractive curve indicate; Categories of tissue: 2 Theta value; and Intensity RV.

OPC CALCULATION FAS vs GAS

∑ DPS + VALUES 94228,17

∑ DPS - VALUES -6385,33

OPC VALUE = (+DPS) / (-DPS) = 14,76

4 FAS vs 2 CAS was analysed. The same phenomena of Glycerolized amnion about form and design maintenance, was observed between FAS and CAS. Equally, there are a maximum peak in FAS d spacing = 3.26 (28.2º in 2θ) and in CAS, the same d spacing. Also a second peak is detectable to FAS in d spacing = 2.36 (40.8º in 2θ) and to CAS with equal

**DIFFRACTOGRAPHIC PROFILES FAS vs CAS**

**MEAN FAS: 40.8; 1940,33**

**MEAN CAS: 40.8; 1302,00**

**MEAN FAS MEAN CAS**

Fig. 9. Diffractive curves FAS and GAS profiles. Note: The 1st and 2nd maximun peak labels

of each respective diffractive curve indicate; Categories of tissue: 2 Theta value; and

Table 1. OPC value from FAS vs GAS.

values. (See Fig 9)

Intensity RV.

**0**

**4**

**6.4**

**8.8**

**11.2**

**13.6**

**16**

**18.4**

**20.8**

**23.2**

**25.6**

**28**

**2 THETA VALUES (O)**

**30.4**

**32.8**

**35.2**

**37.6**

**40**

**42.4**

**44.8**

**47.2**

**49.6**

**52**

**54.4**

**56.8**

**59.2**

**500**

**1000**

**1500**

**2000**

**INTENSITY (R.V)**

**2500**

**3000**

**3500**

**7.3 Cryopreserved amnion tissues results: X-ray diffraction** 

**MEAN FAS: 28.2; 3306,33**

**MEAN CAS: 28.2; 2453,00**

OPERATION ABS. VALUES

Figure 10: show DPS profile obtained from operative subtractions analysis between FAS Vs CAS respective diffractive curves:

Fig. 10. DPS planimetric picture FAS vs CAS.

Operative planimetric values DPS obtained showed an OPC = 42.02 (See Table 2).

Newly highlight the notable differences in shape and form between vascular and amnion DPS pictures.


Table 2. OPC values FAS vs GAS

#### **7.4 Cryopreserved amnion tissues results: Raman Spectra**

Figure 11 Show Raman Spectra profile from FAS vs CAS assays. Note that having regard to the best imaging definition, and taking account the meaningful change area of Raman Shift between both categories, the showed range is 1000 – 2000 cm-1 in *x*.

It should be highlighted that arrows points marked correspond to noticeable differences in positive Intensity values (AU) between FAS (red line) and CAS (blue line).

X Ray Diffraction: An Approach to Structural

CAS, OPC value = 42.02 respectively).

complex vascular structural tissue.

**8.2 Raman scattering** 

tissue categories: cryopreserved amnion and vascular tissue.

Quality of Biological Preserved Tissues in Tissue Banks 457

profiles designs, defined as "molecular crystals". (FAS vs GAS, OPC value = 14.76; FAS vs.

The main conclusion from these data is: amnion chemical glycerolized procedures, change sequencing molecular design, while physical cryopreserved method does not. But, physical cryopreservation method, and chemical glycerolized, modifies OPC values related to both

It must be noted the observed differences in the profiles of diffractive curves between fresh and cryopreserved arterial vessels. Indeed, there are a disparity between those varieties, which were not verified by the corresponding samples fresh and cryopreserved amniotic membrane, that maintain substantially similar profiles. This aspect is independent of the OPC values for both tissues and categories of each study. Our hypothesis is that these differences are related to the anatomical and functional collagen distribution in different tissues. Indeed, the hierarchical order of collagen mesh reach a final design bundles

In this sense, arterial wall of large conduit vessels such as aorta and carotid, are under pulsate hemodynamic regimens alternating expansion and elastic contraction states. Under these conditions, the main loads acting on the vessel wall are pressure and blood flow. The blood pressure acts directly on the inner wall of the vessel in normal direction, and flow proactively work generating a pressure proportional to the square of blood velocity, Fung YC (1997). There are therefore two preferred directions in the distribution of the charges: one circumferential and other longitudinal. This results in a complex morphological organization of the cellular components of the middle layer, composed of smooth muscle cells and collagen mesh ECM, whose design will trace circumferential and longitudinal lines, giving to vascular tissue an anisotropic mechanical behavior condition. (Rodriguez, 2007). The primary ice spontaneous nucleation happen at random in many sites of ECM, Muldrew. 1999). The crystallization growth front, follows the preferred direction lines according to design collagen mesh. According with OPC values, the new organizational picture of the cryopreserved defrosted vascular tissues would be the result of a complex sequence of physic chemical events through preservation procedures, applying changing a

This is not the organizational situation of amnion collagen ECM. Amnion membrane has a laminar design, and is not subject to biomechanical pulsate regimen. Its function as an external fetal covering meets fundamentally amniotic liquid metabolic regulations, more than biomechanical functions. In spite of, both kind of tissue studied, (vascular, and amnion membrane) have basically the same fibril collagen composition, namely: Collagen I, III, and

Then, our hypothesis is that the morphologic compositions, and the architectural organizations, according to specific functional requirements to each tissue, define the proper

Our preliminary results obtained on amnion membrane (FAM vs CAS) show punctual differences between both categories in three ranges of Raman Sepectra: 1260, 1442, and 1667 cm-1 band (See figure 11) where is observed an increased Intensity (AU) values in FAS

V. Additionally, Colagen IV in structural Basements Membranes.

molecular assembly, and therefore its own diffractografic profiles.

arranged following the lines of force according to bio mechanical requirements.

Fig. 11. Raman Spectra FAS vs CAS to area 1000 – 1800 Raman Shift (cm-1). Color code; Red: FAS, Blue: CAS.

#### **8. Comments**

#### **8.1 X-ray diffraction**

The application of XRD on final quality of stromal collagen tissues analyzed, show differential results according tissue type and / or method of preservation applied. Indeed, relative to the observed results in cryopreservation of arterial vessels we see that regardless of the vessel –carotid or descending thoracic aorta- and the condition of FVA or CVS, a common diffractive peak at d spacing 2.86 Å is seen. The same phenomenon is shown for a second peak at d spacing 2.15 Å. These results show that vascular cryopreservation -defrost procedures did not alter the sequential structure of vascular fresh collagen. In reference to the results of amniotic membrane processing under the same preservation procedures, we see that for both varieties, fresh and cryopreserved, the maximum diffractive profiles remain unchanged: FAS, with d spacing 3.262 Å (28.2 ° in 2 θ) and equal values are checked for CAS. Other 2nd peak at d – spacing 2.359 (40.8º in 2 θ) is verified for both study categories. This confirms that the collagen is resistant to the cryopreservation defrost technique in regard to its sequential molecular structure independently of type of tissue.

Contrary, when both amniotic membrane categories study values are observed we found that FAS variant shows a peak for d spacing of 3.241 Å. (28.4 ° in 2θ), while GAS is expressed in the maximum deflection for spacing d 3.284 (28 ° in 2θ). This lag is not verified for the 2nd peak in both categories that match at d – spacing = 2.359 (40.8º in 2 θ). (See figure 7). These findings showed significant data in the sense that the chemical preservation of amniotic membrane with glycerol, modifies sequencing molecular design of collagen, while this variable is not changed under the cryopreserved defrosted condition.

These would be in according with aforementioned work from (Frushour & Koenig 1975).

Additionally, by analyzing the profiles of ordering by OPC values -in relative terms- we see that both, the cryopreservation defrosted and glicerolización procedures, down modify

Fig. 11. Raman Spectra FAS vs CAS to area 1000 – 1800 Raman Shift (cm-1). Color code; Red:

The application of XRD on final quality of stromal collagen tissues analyzed, show differential results according tissue type and / or method of preservation applied. Indeed, relative to the observed results in cryopreservation of arterial vessels we see that regardless of the vessel –carotid or descending thoracic aorta- and the condition of FVA or CVS, a common diffractive peak at d spacing 2.86 Å is seen. The same phenomenon is shown for a second peak at d spacing 2.15 Å. These results show that vascular cryopreservation -defrost procedures did not alter the sequential structure of vascular fresh collagen. In reference to the results of amniotic membrane processing under the same preservation procedures, we see that for both varieties, fresh and cryopreserved, the maximum diffractive profiles remain unchanged: FAS, with d spacing 3.262 Å (28.2 ° in 2 θ) and equal values are checked for CAS. Other 2nd peak at d – spacing 2.359 (40.8º in 2 θ) is verified for both study categories. This confirms that the collagen is resistant to the cryopreservation defrost technique in

Contrary, when both amniotic membrane categories study values are observed we found that FAS variant shows a peak for d spacing of 3.241 Å. (28.4 ° in 2θ), while GAS is expressed in the maximum deflection for spacing d 3.284 (28 ° in 2θ). This lag is not verified for the 2nd peak in both categories that match at d – spacing = 2.359 (40.8º in 2 θ). (See figure 7). These findings showed significant data in the sense that the chemical preservation of amniotic membrane with glycerol, modifies sequencing molecular design of collagen, while

These would be in according with aforementioned work from (Frushour & Koenig 1975).

Additionally, by analyzing the profiles of ordering by OPC values -in relative terms- we see that both, the cryopreservation defrosted and glicerolización procedures, down modify

regard to its sequential molecular structure independently of type of tissue.

this variable is not changed under the cryopreserved defrosted condition.

FAS, Blue: CAS.

**8. Comments** 

**8.1 X-ray diffraction** 

profiles designs, defined as "molecular crystals". (FAS vs GAS, OPC value = 14.76; FAS vs. CAS, OPC value = 42.02 respectively).

The main conclusion from these data is: amnion chemical glycerolized procedures, change sequencing molecular design, while physical cryopreserved method does not. But, physical cryopreservation method, and chemical glycerolized, modifies OPC values related to both tissue categories: cryopreserved amnion and vascular tissue.

It must be noted the observed differences in the profiles of diffractive curves between fresh and cryopreserved arterial vessels. Indeed, there are a disparity between those varieties, which were not verified by the corresponding samples fresh and cryopreserved amniotic membrane, that maintain substantially similar profiles. This aspect is independent of the OPC values for both tissues and categories of each study. Our hypothesis is that these differences are related to the anatomical and functional collagen distribution in different tissues. Indeed, the hierarchical order of collagen mesh reach a final design bundles arranged following the lines of force according to bio mechanical requirements.

In this sense, arterial wall of large conduit vessels such as aorta and carotid, are under pulsate hemodynamic regimens alternating expansion and elastic contraction states. Under these conditions, the main loads acting on the vessel wall are pressure and blood flow. The blood pressure acts directly on the inner wall of the vessel in normal direction, and flow proactively work generating a pressure proportional to the square of blood velocity, Fung YC (1997). There are therefore two preferred directions in the distribution of the charges: one circumferential and other longitudinal. This results in a complex morphological organization of the cellular components of the middle layer, composed of smooth muscle cells and collagen mesh ECM, whose design will trace circumferential and longitudinal lines, giving to vascular tissue an anisotropic mechanical behavior condition. (Rodriguez, 2007). The primary ice spontaneous nucleation happen at random in many sites of ECM, Muldrew. 1999). The crystallization growth front, follows the preferred direction lines according to design collagen mesh. According with OPC values, the new organizational picture of the cryopreserved defrosted vascular tissues would be the result of a complex sequence of physic chemical events through preservation procedures, applying changing a complex vascular structural tissue.

This is not the organizational situation of amnion collagen ECM. Amnion membrane has a laminar design, and is not subject to biomechanical pulsate regimen. Its function as an external fetal covering meets fundamentally amniotic liquid metabolic regulations, more than biomechanical functions. In spite of, both kind of tissue studied, (vascular, and amnion membrane) have basically the same fibril collagen composition, namely: Collagen I, III, and V. Additionally, Colagen IV in structural Basements Membranes.

Then, our hypothesis is that the morphologic compositions, and the architectural organizations, according to specific functional requirements to each tissue, define the proper molecular assembly, and therefore its own diffractografic profiles.

#### **8.2 Raman scattering**

Our preliminary results obtained on amnion membrane (FAM vs CAS) show punctual differences between both categories in three ranges of Raman Sepectra: 1260, 1442, and 1667 cm-1 band (See figure 11) where is observed an increased Intensity (AU) values in FAS

X Ray Diffraction: An Approach to Structural

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related to CAS. According to references Frank, C. et al (1995), these Raman Spectra range areas aforementioned belong to fibril collagen I, III and V from human placenta. The defined corresponding chemical residues assignments, (Frushour & Koenig 1975). are respectively: Amide III; CH3, CH2 (deform); and Amide I for each range Raman Spectra recorded.

These findings support our work hypothesis about the potential power of cryopreservation procedure to change collagen structure at molecular level.
