Structural characterization of four different naturally occurring porcine collagen membranes suitable for medical applications

Abstract

Collagen is the main structural element of connective tissues, and its favorable properties make it an ideal biomaterial for regenerative medicine. In dental medicine, collagen barrier membranes fabricated from naturally occurring tissues are used for guided bone regeneration. Since the morphological characteristics of collagen membranes play a crucial role in their mechanical properties and affect the cellular behavior at the defect site, in-depth knowledge of the structure is key. As a base for the development of novel collagen membranes, an extensive morphological analysis of four porcine membranes, including centrum tendineum, pericardium, plica venae cavae and small intestinal submucosa, was performed. Native membranes were analyzed in terms of their thickness. Second harmonic generation and two-photon excitation microscopy of the native membranes showed the 3D architecture of the collagen and elastic fibers, as well as a volumetric index of these two membrane components. The surface morphology, fiber arrangement, collagen fibril diameter and D-periodicity of decellularized membranes were investigated by scanning electron microscopy. All the membrane types showed significant differences in thickness. In general, undulating collagen fibers were arranged in stacked layers, which were parallel to the membrane surface. Multiphoton microscopy revealed a conspicuous superficial elastic fiber network, while the elastin content in deeper layers varied. The elastin/collagen volumetric index was very similar in the investigated membranes and indicated that the collagen content was clearly higher than the elastin content. The surface of both the pericardium and plica venae cavae and the cranial surface of the centrum tendineum revealed a smooth, tightly arranged and crumpled morphology. On the caudal face of the centrum tendineum, a compact collagen arrangement was interrupted by clusters of circular discontinuities. In contrast, both surfaces of the small intestinal submucosa were fibrous, fuzzy and irregular. All the membranes consisted of largely uniform fibrils displaying the characteristic D-banding. This study reveals similarities and relevant differences among the investigated porcine membranes, suggesting that each membrane represents a unique biomaterial suitable for specific applications.

Fig 1. Box plots of membrane thickness measurements of native CT, PE, PL and SIS.

(A) Membrane thickness was assessed on native membranes (CT, PE, and PL: N = 11; SIS: N = 15). For every sample of CT, PE and PL and for every piece of SIS, three triplicate measurements at three distinct spots were performed. The plica was the thinnest membrane, whereas CT was the thickest. Statistical comparison of the membrane types (Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test) showed that all membrane types were significantly different from each other in terms of median thickness. (B) Box plots show the thickness measurements of all individual samples and pieces for every given membrane type. Statistical analysis showed significant differences within every membrane type. This result illustrates the considerable heterogeneity of the material.

Fig 2. SEM micrographs of porcine collagen membranes.

(A)-(D) show different magnifications (200x to 10,000x) of one sample obtained from the PL. The observed smooth surface morphology of PL is also detectable in PE and on the cranial face of CT. (E)-(H) show micrographs obtained from the smooth side of SIS. (I)-(L) are images obtained from the rough side of SIS, and (M)-(P) depict the magnified micrographs from the peritoneal (i.e., caudal) side of CT. The caudal side clearly shows circular discontinuities (arrows), whereas the cranial side does not (https://doi.org/10.6084/m9.figshare.7082999). (Q) High magnification of fibril bundles reveals the typical periodic banding pattern of collagen fibrils. (R) Collagen fibrils showing right-handed helical grooves (arrows) are occasionally found in all the membrane types.

Fig 3. SHG and TPEF of native porcine membranes.

Micrographs of single optical section planes and 3D composites of z-stacks generated by SHG/TPEF reveal the collagen and elastic fiber arrangement in PL, PE and CT. Collagen is shown in blue, while the elastic fibers appear in green (surface rendered). (A, F, J) are single optical section planes of a superficial tissue layer of the corresponding membrane type. The preferential orientation of the collagen fibers exhibits some variation. Elastic fibers are abundant in the superficial tissue layers. (B, G, K) are single optical section planes in deeper regions of the corresponding membrane type. The collagen alignment is variable again. Elastic fibers in deeper layers are frequently observed in PE and CT but are rare in PL. (C, H, L) are 3D composites of the collagen structure alone, showing undulating collagen fibers arranged in planes parallel to the membrane surface. (D, I, M) are the same 3D composites also showing the elastic fiber network. (E) is a cross-sectional view of the 3D composite of PL, revealing the sandwich-like structure arising from the superficial elastin network and the core of the membrane that is almost devoid of elastin.