7. An approach to molecular characteristic of collagen mesh extracellular matrix in processed tissue banking, by diffractive techniques

Abstract

Listen

Translate article

Preservation techniques are of fundamental support for conservation of tissue banking (TB) biological materials. However, controversial views remain on the effects at the molecular level that preservation procedures could produce on the functional structures of tissues. Both programmed cryopreservation and glycerolized tissue preservation are common TB methods in technical preservation. Methodological analysis must be introduced in order to find structural differences in the basic material tissue constitutive collagen to compare potential changes between different procedures. To evaluate comparative final characteristics of programmed cryopreserved vs. glycerolized amnion related to its respective extra cellular matrix (ECM) collagen mesh, using X-ray, and Raman scattering diffractive techniques. Six amnion donors (mean age: 24.33 ± 5.37) were selected according to validated International Standards, informed consent, and clinically controlled normal pregnancy and delivery in accordance with the Ministry of Public Health of Uruguay. Under aseptic conditions amnion tissue was manually dissected from each placenta. Six amnion samples were taken as control: fresh amnion (FAM), stored in saline solution for 24 h, and shipped to DETEMA for diffractographic analysis. Six samples were glycerolized (95%) (GAM) and stored at 4 °C for 30 days. Two amnion samples were processed for cryopreservation (CAM) with RPMI 1640 (90%) and Me 2 SO (10%), and freezing at a controlled rate (1 °C/min) until −142 °C, and stored for 30 days at the same temperature. 30 day amnion samples GAM and CAM, were then shipped for the same diffractographic assays. Defrosting protocol applied to CAM was in accordance with Pegg et al. (Cryobiology 34 (1997) 183–192). Diffractographic analysis was applied and compared with respective profile curves: mean FAM values vs. mean GAM and CAM values calculating OPC values according with Perez Campos et al. (Transplantation Proceedings 40 (2008) 668–674). According to Bragg’s Law, the comparative distance between the planes of the crystalline design of collagen mesh (d space) was calculated relating to each procedure vs. FAM. Additional Raman scattering comparative considerations were obtained. Both GAM and CAM diffractographic curves had the same design related to FAM. However, respective OPC values (FAM vs. GAM = 14.76; and FAM vs. CAM = 42.02) show a higher relative collagen mesh ordering in GAM respective CAM comparing with control FAM. But when comparing the maximum diffractive peak GAM and CAM vs. FAM there was a mismatch with GAM samples of 0.4° in the incidental X-Ray angle: two theta values ( θ ) of Bragg’s Law. On the contrary no mismatch was observed in CAM samples related with FAM. Comparative Raman spectra profiles between FAM and CAM showed punctual differences at ranges of 1260, 1442, and 1667 cm −1 where increased relative intensity values in FAM related to CAM were observed. Conclusions: X-ray diffraction profiles show higher relative ordering of GAM related to CAM but glycerolization modified the structural crystalline design of collagen mesh. Cryopreservation modifies fibril collagen of human amnion I, III and V in chemical residues measured with Raman spectra technique, according to Frank, C. & McCreey, R. (Anal. Chem. 67 (1995) 777–783), and Frushour & Koenig. (Biopolymers 14 (1975) 363–377).