**5. Diffractometry: a tool for analysis of structural ordering of collagen**

### **5.1 The matter and its organization**

It defines that the spatial arrangement of ions and atoms of matter, have a crystalline profile when its design shows a repeating sequence. The frequency of repeated and symmetrical distribution of the atomic stereo chemical units constituents of matter, determine the solid crystalline character. It is understood that a substance is "homogeneous" when each constituent unit of the solid is linked by chemical bonds to another identical unit in any sense of space, and is identified as the ideal "homogeneous crystal" model when, theoretically, is infinitely extended in space. The sequential nature of the repetitive and symmetric atomic elements, defines the spatial network model, under the so called "cells ordering". The three dimensional symmetric distribution of elemental units of the complex let likened to an orderly succession of planes separated by a distance "d". This design is easily identifiable in crystalline substances of inorganic chemistry: quartz, diamond, graphite, etc. Diffracted analysis of inorganic or organic crystalline matter can provide detailed information about molecular design, related to intermolecular distance and stereo chemical angle conformation. In the world of bio molecular chemistry, matter is organized by more complex models, through an extensive variety of atomic molecular combined structures. In this picture certain combinations become repetitive units, consisting of several basic types of atoms links together by different kinds of bonds. Nevertheless, one can observe the character of certain spatially ordered molecular configurations, which are equally repetitive. This setting defines the so called "molecular crystals", despite not

X Ray Diffraction: An Approach to Structural

**5.3 The Raman effect, or inelastic scattering of light** 

Fig. 1.

**INTENSITY R.V**

**4**

**6.6**

**9.2**

**11.8**

**14.4**

**17**

**19.6**

**22.2**

**24.8**

**27.4**

**30**

**2 THETA VALUES(O)**

**32.6**

**35.2**

**37.8**

**40.4**

**43**

**45.6**

**48.2**

**50.8**

**FAS**

**53.4**

**56**

**58.6**

units (R.U.) (See Figure 2)

Quality of Biological Preserved Tissues in Tissue Banks 447

**DIFFRACTOGRAPHIC PROFILE FRESH AMNION SAMPLE: FAS**

Given a monochromatic light beam incident on a material, there will be a phenomenon of elastic light scattering as a result of the interaction of photons and electron atomic elements of the network links. The elastic scattering implies that the frequency of the incident light beam and the scattered light emerging is the same, so that no changes have occurred in the respective energy levels. This phenomenon known as Rayleigh scattering is highly significant from a statistical point of view. However an extremely low intensity incident light (in the order of 1 photon in 107 to 1010) shows inelastic behavior in the interaction with the structure determining slight changes in the emerging wave frequency, which depends on the characteristics of matter incised. This phenomenon or Raman Effect was discovered by (Raman & Krishnan, 1928), and allows the chemical structure analysis of biological material. Atomic particles mass and its energy states (vibration and/or rotational), maintain chemical bonds that define crystal structure, sustaining dynamic design stability by neighbor interaction. The movements of both vibration and rotation of the particulates: (υ = frequency), defined the dynamically stable energy level where they are. If the interaction of a beam of photons of frequency (υ**0)**, print a change of unstable frequency at the particle network link, they scatter photons with a different frequency (υ**r**), define an inelastic scattering. The energy can be dispersed in a model υ**r** > υ**0** called scattering Raman - Stokes or υr < υ0 know as scattering Raman anti Stokes. υ**r** values are characteristic of each design structures and define atomic molecular matter. Approximately 99% of the output assays is Stokes Raman scattering hence, those are the models profiles recorded. In the Cartesian co ordinate system the independent variable *x* records the difference **υr-υ<sup>0</sup>** in cm**-1**, and the ordinates *y,* the scattering intensities for each differential rate, in relative

showing the perfection system of "homogeneous crystals". So, no bond lengths and angular atomic positions can be determined, but an approximate view about relative ordering structure is given applying XRD techniques. These structures are typical of biological substances such as proteins or DNA molecules whose functional design depends on the molecular arrangement, and type of chemical bond established between its molecules.

#### **5.2 The diffractive phenomenon and elastic scattering of x-rays**

X-rays are a form of electromagnetic energy produced by a source to be impacted by electrons of high kinetic energy supplied (usually a tungsten filament named cathode). The incident electron impact, destabilizes the internal atomic orbital of a target material (an anode built with pure copper), generating atoms in electronically excited state. The movement of electrons from outer orbital to balance the impact generates heat energy and emission of x-rays, in a spectrum of wavelengths (λ) measurable in Angstrom units (**1 Å = 10-10 m**). The band spectral x-rays emission is "filtered" through mono chromator to obtain a single wavelength that corresponds to the "characteristic radiation" used in the x-rays crystallography: K-alpha line (Kα) for each material property anode of the device. To our work, XRD is defined by the interference between monochromatic characteristic emissions (Kα) with the ordered material in crystalline form. When a photon interferes with an orderly molecular structure without loss of energy (elastic scattering), produces a deviation from its original direction which is the diffraction phenomenon. The condition for diffraction to be possible is that the distance between the periodic and ordered structure elements (atoms or molecules), fall in the wavelength range of incident ray. The condition to be detectable is that some degree of ordering is present in the material to be studied, in order that the interference could be constructive. As previously mentioned, collagen shows particular characteristics like a biomolecular arrangement built by: a) the hierarchical supra molecular order fibers, b) the observed crystalline structure of the spaces sequenced "D" of fibrillar collagen, c) the nano scale distance of repetitive molecular units, all of them allows to analyze biological stroma collagen by the XRD. (Aspden, 1987; Berenguer, 2009; Connon, 2007; Hickey & Hukins, 1980; Horton, 1958; Pauling & Corey, 1951; Pérez Campos, 2008).

The equation that allows the practical application of this technique is Bragg's Law.

$$\mathbf{n} \; \lambda \mathbf{\hat{z}} = 2 \mathbf{d} \; \sin \theta \; \tag{1}$$

Formula 1 Bragg´s Law**: n** is an integer**, λ** is the x – ray monochromatic wavelength, d is de distance between the planes of the crystalline net, and **θ** is the angular value between the incidental x –ray and the considered crystalline plane**.** 

Given a certain incidence angle of a monochromatic beam on the material structure, sequencing crystalline spacing "d" determines a dispersive interference when the rays are emerging in construction phase. Emerging rays can be recorded in a Cartesian coordinate system where the independent variable "x" records the range of 2θ values and the dependent variable "y", the relative values (R.V.) of the ordering lattice system. The recordable diffraction graphics or diffractogram depend of the content and the atomic distribution within the repetitive units that define the three dimensional arrangement. (See Figure 1)

Fig. 1.

446 Current Frontiers in Cryopreservation

showing the perfection system of "homogeneous crystals". So, no bond lengths and angular atomic positions can be determined, but an approximate view about relative ordering structure is given applying XRD techniques. These structures are typical of biological substances such as proteins or DNA molecules whose functional design depends on the molecular arrangement, and type of chemical bond established between its molecules.

X-rays are a form of electromagnetic energy produced by a source to be impacted by electrons of high kinetic energy supplied (usually a tungsten filament named cathode). The incident electron impact, destabilizes the internal atomic orbital of a target material (an anode built with pure copper), generating atoms in electronically excited state. The movement of electrons from outer orbital to balance the impact generates heat energy and emission of x-rays, in a spectrum of wavelengths (λ) measurable in Angstrom units (**1 Å = 10-10 m**). The band spectral x-rays emission is "filtered" through mono chromator to obtain a single wavelength that corresponds to the "characteristic radiation" used in the x-rays crystallography: K-alpha line (Kα) for each material property anode of the device. To our work, XRD is defined by the interference between monochromatic characteristic emissions (Kα) with the ordered material in crystalline form. When a photon interferes with an orderly molecular structure without loss of energy (elastic scattering), produces a deviation from its original direction which is the diffraction phenomenon. The condition for diffraction to be possible is that the distance between the periodic and ordered structure elements (atoms or molecules), fall in the wavelength range of incident ray. The condition to be detectable is that some degree of ordering is present in the material to be studied, in order that the interference could be constructive. As previously mentioned, collagen shows particular characteristics like a biomolecular arrangement built by: a) the hierarchical supra molecular order fibers, b) the observed crystalline structure of the spaces sequenced "D" of fibrillar collagen, c) the nano scale distance of repetitive molecular units, all of them allows to analyze biological stroma collagen by the XRD. (Aspden, 1987; Berenguer, 2009; Connon, 2007; Hickey & Hukins, 1980; Horton, 1958; Pauling & Corey, 1951; Pérez Campos,

The equation that allows the practical application of this technique is Bragg's Law.

incidental x –ray and the considered crystalline plane**.** 

 n= 2d sin Formula 1 Bragg´s Law**: n** is an integer**, λ** is the x – ray monochromatic wavelength, d is de distance between the planes of the crystalline net, and **θ** is the angular value between the

Given a certain incidence angle of a monochromatic beam on the material structure, sequencing crystalline spacing "d" determines a dispersive interference when the rays are emerging in construction phase. Emerging rays can be recorded in a Cartesian coordinate system where the independent variable "x" records the range of 2θ values and the dependent variable "y", the relative values (R.V.) of the ordering lattice system. The recordable diffraction graphics or diffractogram depend of the content and the atomic distribution within the repetitive units that define the three dimensional arrangement. (See

**5.2 The diffractive phenomenon and elastic scattering of x-rays** 

2008).

Figure 1)

#### **5.3 The Raman effect, or inelastic scattering of light**

Given a monochromatic light beam incident on a material, there will be a phenomenon of elastic light scattering as a result of the interaction of photons and electron atomic elements of the network links. The elastic scattering implies that the frequency of the incident light beam and the scattered light emerging is the same, so that no changes have occurred in the respective energy levels. This phenomenon known as Rayleigh scattering is highly significant from a statistical point of view. However an extremely low intensity incident light (in the order of 1 photon in 107 to 1010) shows inelastic behavior in the interaction with the structure determining slight changes in the emerging wave frequency, which depends on the characteristics of matter incised. This phenomenon or Raman Effect was discovered by (Raman & Krishnan, 1928), and allows the chemical structure analysis of biological material.

Atomic particles mass and its energy states (vibration and/or rotational), maintain chemical bonds that define crystal structure, sustaining dynamic design stability by neighbor interaction. The movements of both vibration and rotation of the particulates: (υ = frequency), defined the dynamically stable energy level where they are. If the interaction of a beam of photons of frequency (υ**0)**, print a change of unstable frequency at the particle network link, they scatter photons with a different frequency (υ**r**), define an inelastic scattering. The energy can be dispersed in a model υ**r** > υ**0** called scattering Raman - Stokes or υr < υ0 know as scattering Raman anti Stokes. υ**r** values are characteristic of each design structures and define atomic molecular matter. Approximately 99% of the output assays is Stokes Raman scattering hence, those are the models profiles recorded. In the Cartesian co ordinate system the independent variable *x* records the difference **υr-υ<sup>0</sup>** in cm**-1**, and the ordinates *y,* the scattering intensities for each differential rate, in relative units (R.U.) (See Figure 2)

X Ray Diffraction: An Approach to Structural

Health of Uruguay.

up to 30 days at -142ºC.

analysis.

sec each.

and 3400 cm-1 region.

Quality of Biological Preserved Tissues in Tissue Banks 449

Spanish Association of Tissue Banks (AEBT - 2005). The same selection criteria, exclusion, tissue procurement and processing were applied to living donor placenta with clinical controlled normal pregnancy and delivery, by the parameters set by the Ministry of Public

Donors were selected according to the protocol in a range between 18 and 60 years (35.5 ± 11.8 years mean age, 47% M 53% F) obtained by aseptic dissection, 10 aortic arterial segments, and 8 carotid. It proceeded under a laminar flow cabinet to cleaning, package, and storage in physiological saline at 4°C. Fresh Vascular Samples (FVS) were shipped to DETEMA within 24 hs, for XRD. The same process aseptic protocol was applied to 6 amnion obtained by manual dissection from donor placenta. Fresh Amnion Samples, (FAS) were stored in saline solution at 4ºC and shipping to DETEMA within 24 hs for XRD

The Cryopreserved Vascular Samples (CVS), segments of each contra lateral carotid donor, and hemi ring segments of thoracic descendent aorta, were processed for cryopreservation in a Controlled Rate Freezing System (Model 9000, Gordinier Electronics, Inc. Michigan). Stored CVS were maintained up to 30 days at -142ºC. The cryopreservation media was: RPMI 1640, 85 cc; Human Albumin (20%), 5 cc; DMSO 10%. Cryopreservation was made into termal sealed double cryo resistant bag (Joisten and Kettenbaum D51429, Bereisch Gladbach, Mod.011342). The mean cooling rate applied was -1ºC/min from 4ºC to -90ºC and

Same procedures were applied to obtain 2 Cryopreserved Amnion Samples (CAS), stored

Defrost protocol applied for vascular and amniotic tissues, was according with Pegg et al.

6 Glycerolized Amnion Samples (GAS) were obtained by soaked in screw cap flask in Glycerol (95%) and stored at 4º C for 30 days. Glycerol from amnion was removed by three sequential shaking washing for 15 min. each, in saline solution and then shipped to DETEMA for x-rays diffraction, and Raman Scattering analysis. The comparative assays

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

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

then quickly stored at -142ºC during 30 days in steam liquid nitrogen.

(1997), and defrosted samples were shipped to DETEMA for XRD.

were done with FVS vs CVS; FAS vs CAS; and FAS vs GAS.

**6.2 Diffractographic and Raman scattering technical procedures** 

Fig. 2. Raman Spectum oleic acid

#### **5.4 Interaction x-rays - Collagen: A model analysis**

The x-rays diffractive analysis of the collagen structure was earlier studied (Pauling & Corey, 1951). Defined diffraction peak was described, in the range d = 2.86 Å (2 θ = 31.3 º using CuK α radiation λ = 1.5418 Å value), based on the criteria of the Bragg Law. This phenomenon was interpreted as the expression of cis configurations for the amide groups of the polypeptide chain.

Using this background, our group analyzed tissue banking allograft, in order to compare the diffraction profiles obtained before and after cryo-preservation method (vascular tissues and amnion tissue), and glycerolized preserved method (amniotic membrane). The working hypothesis states that the preservation methods can modify the stereochemistry molecular structures, determining changes in collagen and the consequent differentiation of diffractive or dispersive profile.
