**3. The concept of functional structure of ECM**

The ECM was considered early in the twentieth century as filler material and mechanical support of cell structures, which was thought as the only protagonists of tissues functionality. In the 50´s, (Grobstein, 1953) proposed that the induction in the development of a tissue depended on the presence of ECM.

In the 60´s, (Hauschka & Königsberg, 1966) establish that pig embryonic muscle cell cultures proliferate more properly in a media with presence of metabolic products from fibroblasts, and identify the collagen to induce the development of them. This protagonist role of collagen in the processes of induction and cell proliferation, is corroborated by (Meier & There, 1974), by testing the inductive capacity of collagen, for the synthesis of ECM in the corneal epithelium of pig embryos (5 days old development). (Sanders 1988) working on neural crest and sclerotome cells of early chicken cells embryos, prove that the presence of type I collagen is necessary for cellular migration and *de novo* synthesis of ECM is a prerequisite for normal cell migration and attachment in earliest stages of embryogenesis.

Additionaly, since the early 80´s (Bissell 1982), work in the field of cancer biology emphasizes the importance of "micro" immediate cellular environment and posits the hypothesis of 'Dynamic Reciprocity' by which the ECM contact trans membrane receptors, influence gene expression through signals transmitted via cytoskeleton, generating so "new" products for the ECM. Thus, the cell and the ECM, form a binomial reciprocal exchange interaction, which has vital importance in the early stages of embryonic morphogenesis and later in postnatal life, the physiological mechanisms of growth and development as well as in response to injury. (Bissell, 1982; Davis, 2010; Kelleher, 2004; Nadiarnykh, 2010; Schultz, 2005; Schwinn, 2010). The ECM consists of a complex variety of macro molecules that can be summarized schematically as follows: 1) protein collagen, 2) structural glycoproteins, 3) proteoglycans and glucosamine glycans, and 4) elastin. These complex and diverse

their ethical rules. On the other hand, tissue banking activities are carried out following strict bio-security criteria by the selection of suitable donors, permanent quality control and continuous management of physical plant, equipment, supplies procurement procedures,

According to (Pegg 2006) this concept of "viability," applies whenever a graft of tissue obtained as final product, meets the leading natural-biological function for which it was preserved, and that is pathologically affected in the recipient. This explains why there are different procedures and methods of conservation in TB, according to the type of tissues and

In some cases, we must preserve, as the main function, a biological synthesis, which requires mandatory of vitality in cellular functions (eg, parathyroid glands, or ovarian tissue). In other cases, the objective is to preserve static and mechanical functions as a segmental allograft bone support, or biodynamic behavior, as in cases of preservation of blood vessels. This implies that "viability" is not necessarily synonymous with "cell vitality" and therefore

Hence, the importance of assessing possible changes on the components of the ECM that TB

The ECM was considered early in the twentieth century as filler material and mechanical support of cell structures, which was thought as the only protagonists of tissues functionality. In the 50´s, (Grobstein, 1953) proposed that the induction in the development

In the 60´s, (Hauschka & Königsberg, 1966) establish that pig embryonic muscle cell cultures proliferate more properly in a media with presence of metabolic products from fibroblasts, and identify the collagen to induce the development of them. This protagonist role of collagen in the processes of induction and cell proliferation, is corroborated by (Meier & There, 1974), by testing the inductive capacity of collagen, for the synthesis of ECM in the corneal epithelium of pig embryos (5 days old development). (Sanders 1988) working on neural crest and sclerotome cells of early chicken cells embryos, prove that the presence of type I collagen is necessary for cellular migration and *de novo* synthesis of ECM is a prerequisite for normal cell migration and attachment in earliest stages of embryogenesis.

Additionaly, since the early 80´s (Bissell 1982), work in the field of cancer biology emphasizes the importance of "micro" immediate cellular environment and posits the hypothesis of 'Dynamic Reciprocity' by which the ECM contact trans membrane receptors, influence gene expression through signals transmitted via cytoskeleton, generating so "new" products for the ECM. Thus, the cell and the ECM, form a binomial reciprocal exchange interaction, which has vital importance in the early stages of embryonic morphogenesis and later in postnatal life, the physiological mechanisms of growth and development as well as in response to injury. (Bissell, 1982; Davis, 2010; Kelleher, 2004; Nadiarnykh, 2010; Schultz, 2005; Schwinn, 2010). The ECM consists of a complex variety of macro molecules that can be summarized schematically as follows: 1) protein collagen, 2) structural glycoproteins, 3) proteoglycans and glucosamine glycans, and 4) elastin. These complex and diverse

and end products, which must comply criteria for "viability" therapeutic apply.

the requirement is to preserve elements of the Extra Cellular Matrix (ECM).

procedures may be generate on processing and stocking biological tissues.

**3. The concept of functional structure of ECM** 

of a tissue depended on the presence of ECM.

its expected restored function.

molecular groups, organized into super families, are shown with a dynamic distribution, and functional modulated behavior, with variations between different tissues. This bio plasticity is observed even within the same tissue type, as homeostatic biochemical, and bio mechanical requirements, including interactions with various molecules: growth factors, cytokines, enzymes and other inducers synthesis products as well as lytic and degradative matrix one.

The surface receptors of the cell membrane, in close contact with this complex and dynamic molecular ECM set, interact through the cytoskeleton to the genome, which modulates the different stages of ontogeny, growth and postnatal development sequences as well as molecular structural and functional physiology and patho physiology biological requirements of tissues. (Abraham, 2007; Bowers, 2010; Worthley, 2010). Related to the own collagen structure there are ligands and functional domains that take contact with other ECM molecules (fibronectin, proteoglicans, and collagen - collagen interactions) and with the neighbor cells microenvironment (cell integrin receptors). Several poly peptid sequences, (eg.: GFPGER: glycine - phenylalanine - hyroxyproline - glycine - glutamic acid - arginine) and ligands domains (eg.: Matrix Metalloproteinase Interaction Domain or MMP ID; Colagen V Cross-link site or Col V X-link), have been identified and play an important role in regulation of migration, proliferation, adhesion and apoptosis in biology cell tissues. (Orgel, 2011; Sottile, 2007; Sweeney, 2008).

It is obvious therefore that the extra cellular medium, forms a molecular complex of plastic, in both dynamic up regulator as down regulator in constant cross talk with cell pole whose points of contact and mutual information imply the presence of binding sites at ECM structure related to cell surface receptors. Fibril collagen constitutes approximately 25% of tissues for all species of mammals and is the main component of the total molecules that make up the ECM. (Kielty, & Grant, 2002). To date, they have been described up to 29 different types of collagens with the corresponding genetic determinants. "Structural" Collagens in the ECM are called fibrils (Fibril Collagen) consisting of types I, II and III, V and XI. Type I constitute 90% of body collagen and mainly, perform mechanical resistance functions. In addition it provides three - dimensional modeling formation of tissues. An important bio molecular feature of our study is it hierarchical and sequenced shaping showing collagen. Taking collagen I model, pro collagen, amino acid primary structure have a intracellular synthesis (endoplasmic reticulum), with repeated tripeptides, design whose residues are Gly-Pro- Hyp or Gly-X- Hyp which Pro and Hyp are near to 17% and 33% respectively. Therefore 50% of the average 1000 residues of the total composition of the molecule, pro collagen, are other amino acids. Its length of 300 nm and width of 1.5 nm, is organized in a left-handed secondary structure of three amino acids per turn, with Gly residues central and peripheral Pro and Hyp out of the spiral. Three assembly helical pro collagen monomers (2 α1 and 1 α2) in right-handed configuration, determine the tropo collagen structure in the extra cellular space. In this space the molecule is arranged in staggered bundles with a gap of 67 nm by inter molecular bonds tropo collagen units, which gives to the collagen fibril new product design, a repetitive sequence which observed in the ME identified a characteristic D - banding. The final design shows collagen fibers arranged, spatially distributed in regular packages along the lines of force of the biomechanical characteristics of each tissue. This last aspect brings an added dimension of ordering design given by the spatial distribution of fiber bundles, and their inter reciprocal space.

X Ray Diffraction: An Approach to Structural

Park, 2009) or bone tissues. (Hamer, 1996).

cryopreservation defrosted process in TB.

**5.1 The matter and its organization** 

the hydrophobic bonds.

Layland, 2007).

Quality of Biological Preserved Tissues in Tissue Banks 445

fresh and cryopreserved human heart valves by second harmonic generation, sets no changes between both categories. However, it was found extensive damage in collagen structure in porcine frozen leaflets related to fresh control one, using laser-induced auto fluorescence imaging , (Schenke Layland, 2006), and second-harmonic generation. (Schenke

In other field, there are many studies showing that the biomechanical behavior of tissue collagen framework, is not altered by effect of cryopreservation / thaw cycle, in vascular (Armentano, 2006; Bia, 2006; Langerak, 2001, 2007; Pukacki, 2000**),** tendon , (Woo, 1986;

However, controversial literature is also observed for biomechanical variables. (Rosset, 1996) observed in vitro, decreased compliance and hysteresis an increase of modulus of elasticity in thawed cryopreserved human carotid arteries, related to fresh one. (Gianni, 2008) found that the freezing of human posterior tibial tendons significantly affected behavior in vitro biomechanical performance. Finally, either way, under many point of view is possible highlight that the functional character of fibril collagen depending to the particular structural and biochemical preservation, which may be damaged during

About the interaction between alcohols and polymerized amino acid, in early 70´s (Frushour, & Koenig, 1975) postulated, in Raman Scattering field, that methanol modified an aqueous poly-DL-alanine (PDLA) solution, by disruptions of the helical regions by breaking

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

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

These three characteristics: a) repetitive and periodic sequencing of the D - banding, b) structuring hierarchically ordered by supra molecular complexes, and c) nano-scale dimensions of the structures, made of collagen complex an para crystalline super molecule, liable to be analyzed by techniques diffractive as discussed later. (Sweeney, 2008; Berenger, 2009).

In this context becomes important the analysis of the changes that for preservation purposes, can be induced in the molecular components of the ECM. Particularly taking account that collagen is the main structural component of the extra cellular microenvironment and has a proven role in the functional biological mechanisms, developmental, physiological homeostatic and physio - pathological tissues behavior. (Kielty & Grant, 2002; Orgel, 2011).

These reasons justify work TB, to design tissue preservation processing models, with conservation of the collagen component, from both, structural and biochemical characteristics. It must be take in mind, that allografts should meet a homeostatic interaction with cell biology recipient patient, through its membrane receptors in functional contacts with molecular ligands and domains of preserved ECM, to improve physio pathological situations.

Note therefore that the biological behavior of a suitable allograft depend on the presence and indemnity of molecular epitopes or ligands, which can be eventually altered in its stereo chemical distribution during cryogenic or glycerolized procedures.
