Atomic force microscopy investigation of chemically stabilized pericardium tissue

Abstract

Native and chemically stabilized porcine pericardium tissue was imaged by the contact mode atomic force microscopy (AFM), in air. Chemically stabilized pericardium is used as a tissue-derived biomaterial in various fields of the reconstructive and replacement surgery. Collagen type I is the main component of the fibrous layer of the pericardium tissue. In this study, the surface topography of collagen fibrils in their native state in tissue and after chemical stabilization with different cross-linking reagents: glutaraldehyde (GA), dimethyl suberimidate (DMS) and tannic acid (TA) was investigated. It has been found that chemical stabilization causes considerable changes in the surface topography of collagen fibrils as well as in the spatial organization of the fibrils within the tissue. The observed changes in the D-spacing pattern of the collagen fibril correspond to the formation of intrafibrilar cross-links, whereas formation of interfibrilar cross-links is mainly responsible for the observed tangled spatial arrangement of fibrils and crimp structure of the tissue surface. The crimp structure was distinctly seen for the GA cross-linked tissue. Surface heterogeneity of the cross-linking process was observed for the DMS-stabilized tissue. SDS-PAGE electrophoresis was performed in order to evaluate the stabilization effect of the tissues treated with the cross-linking reagents. It has been found that stabilization with DMS, GA or TA enhances significantly the tissue resistance to SDS/NaCl extraction. The relation between the tissue stability and changes in the topography of the tissue surface was interpreted in terms of different nature of cross-links formed by DMS, GA and TA with collagen.

Tannic acid‐stabilized pericardium tissue: IR spectroscopy, atomic force microscopy, and dielectric spectroscopy investigations

“…The observed changes in surface topography of the TA‐stabilized tissue can support formation of both multiple intra‐ and interfibrilar TA–collagen crosslinks. These results are in good agreement with those of earlier AFM investigations performed for chemically stabilized tissues 25…”

Tissue section AFM: In situ ultrastructural imaging of native biomolecules

“…Where AFM of sections has been reported, the tissues had been previously exposed to fixation and embedding procedures (Li et al, 2008; Matsko and Mueller, 2004). The impact of such chemical fixation protocols on protein structure was, however, recognised in the early 1950s (Fernandezmoran, 1952) and confirmed in recent AFM studies of bulk tissue samples (Jastrzebska et al, 2005). Consequently, tissue freezing and cryo-sectioning, now widely utilized in immuno-cytochemical studies (Akagi et al, 2006), were developed to prevent protein denaturation and larger-scale alterations in structural morphology.…”

Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and Label-Free Imaging Techniques

“…On the other hand, scanning probe microscopy techniques, such as atomic force microscopy (AFM), provide superior resolution to conventional optical methods, and in fact, it has been used to monitor the D-spacing pattern in GA cross-linked pericardium, where changes in the D-spacing were observed during the formation of interfibrillar cross-links . Furthermore, Young’s modulus of collagen tissues can be estimated from AFM force–distance curves. , Hence, AFM could be used to investigate the nanostructure of GE cross-linked nanocomposites, where the surface images revealed highly compact structures. , In particular, multiparametric AFM investigations can ideally complement optical data to monitor environmental influences …”