**4. The interaction between ECM components, and the physic-chemical phenomena preservation procedures**

About the cryogenic effects on ECM, there are several references in different disciplines, about the ultra cold temperatures on biological material. Indeed, at the molecular level have been observed different types alterations generated by freezing / thawing phenomenon, on biological structures. As earlier in the 60´s, (Levit, 1962; 1966), had postulated irreversible changes in the tertiary structures of soluble vegetal proteins, with loss of its biological capacity, such as the rearrangement of disulfidric functional bonds to non-functional disulfide covalent configurations.

 The concept of "repulsion hydration forces" refers to cell membranes, was developed by Wolfe, J. (1999). These phenomena is induced by water efflux through such semi-permeable membranes during cooling process, promoting large mechanical stress and strain in the biological structures, and generating physical deformations and changes in the molecular functional membrane behavior. Such condition is done under the observed ground of the structure of the crystalline ice mass in the extra cellular space. This, results in intra cellular dehydration of the tissue and the extra cellular hyper osmolarity of super cooling liquid. So, it generates a displacement of inter atomic and molecular chemical equilibrium that, explains changes in the stereochemistry and molecular architecture of biological structures. This scope would agree to Levit postulates. (Levit, 1962; 1966).

The effect that the conventional criopreservation exerts on the ECM structure is controversial information. (Gerson, 2009), comparing morpho structural collagen mesh from

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,

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.

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

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

About the cryogenic effects on ECM, there are several references in different disciplines, about the ultra cold temperatures on biological material. Indeed, at the molecular level have been observed different types alterations generated by freezing / thawing phenomenon, on biological structures. As earlier in the 60´s, (Levit, 1962; 1966), had postulated irreversible changes in the tertiary structures of soluble vegetal proteins, with loss of its biological capacity, such as the rearrangement of disulfidric functional bonds to non-functional

 The concept of "repulsion hydration forces" refers to cell membranes, was developed by Wolfe, J. (1999). These phenomena is induced by water efflux through such semi-permeable membranes during cooling process, promoting large mechanical stress and strain in the biological structures, and generating physical deformations and changes in the molecular functional membrane behavior. Such condition is done under the observed ground of the structure of the crystalline ice mass in the extra cellular space. This, results in intra cellular dehydration of the tissue and the extra cellular hyper osmolarity of super cooling liquid. So, it generates a displacement of inter atomic and molecular chemical equilibrium that, explains changes in the stereochemistry and molecular architecture of biological structures.

The effect that the conventional criopreservation exerts on the ECM structure is controversial information. (Gerson, 2009), comparing morpho structural collagen mesh from

**4. The interaction between ECM components, and the physic-chemical** 

chemical distribution during cryogenic or glycerolized procedures.

This scope would agree to Levit postulates. (Levit, 1962; 1966).

2009).

situations.

(Kielty & Grant, 2002; Orgel, 2011).

**phenomena preservation procedures** 

disulfide covalent configurations.

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 Layland, 2007).

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

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 cryopreservation defrosted process in TB.

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 the hydrophobic bonds.
