Tuesday, April 19, 2016

Marine-derived collagen biomaterials from echinoderm connective tissues

Echinoderms are marine organisms such as sea stars, sea urchins, and sea cucumbers. They possess connective tissues referred to as mutable collagenous tissues (MCTs). These MCTs have the potential to be developed into collagen barrier-membranes by guided tissue regeneration (GTR) for the use of humans. This study compared echinoderm-derived collagen membranes (EDCMs) to commercially used membranes from bovine collagen substrates (BCMs). Traditional commercial membranes have several areas where they lack causing the search for alternative sources. They can cause allergic reactions, conflict with religious beliefs, have disease transmission connected reasons, and they have a high cost of recombinant technologies. Echinoderm MCTs are relatively easier to obtain high amounts of native fibrils which maintain their original structure, this rapidly produced fibrillar collagen gives high similarities in terms or ultrastructural and mechanical characteristics to the physiological prestige of connective tissue.

Echinoderm Tissues
This study optimized different extraction techniques to efficiently obtain clean, relatively pure and highly concentrated native collagen fibril suspensions from three echinoderm MCTs species. The species studied were the sea urchin Paracentrotus lividus, the sea star Echinaster sepositus, and the sea cucumber Holothuria tubulosa; all of which differ in overall collagen fibril, fiber organization, and the presence of skeletal elements. Sea star aboral arm wall and partly sea urchin peristomal membrane showed highly packed fibrils and calcareous ossicles, where sea cucumber body wall contains loosely packed and wide spread fibrils and small calcareous spicules. This explains why fibril extraction is easily obtained by mild non-denaturing methods for sea cucumber and stronger treatments for both sea urchin and sea star. All three species showed the ability to maintain fibrillar conformation and integrity throughout the extraction process which is promossing compared to less stable mammalian collagen.

Collagen Network of Different Membranes
Glcosaminoglycan (GAG) is a fundamental substance to maintain fibril integrity by cell migration, adhesion, proliferation, and differentiation. GAG is often added to mammalian collagen scaffolds to improve their performances in tissue engineering applications. EDCMs already contain GAG in their native fibrillar collagen. Average porosity of EDCMs was smaller than the size of human cells, allowing them to likely be more efficient as cell barriers for biomedical applications where division of two anatomical compartments is needed such as GTR. In GTR these barriers help the healing process avoiding mixture of adjacent regenerating tissues and are useful to prevent post-surgical tissue adhesions which are common surgical complications.

EDCMs also outperform mammalian collagen due to their limited thickness, high mechanical resistance, and hadleability during surgery. The higher the tensile strength and the resistance to uni-axial tension the better the biomaterial can support stresses before rupture. The mechanical resistance of thicker bovine-derived collagen membranes is much lower compared to EDCMs especially in mechanically demanding tissue engineering application, suggesting the potential utility of echinoderm collagen.

In vitro tests with human skin-derived fibroblasts were conducted to see if cells seeded on the substrates were viable and able to actively proliferate in long-term periods of 21 days. Cells seeded to EDCMs and BCMs were similar in morphology, organization, and substrate adhesion pattern. EDCMs presented a more elongated shape and less and shorter filopodial processes. This can indicate a reduction in substrate adhesion which can be beneficial in cell barrier for GTR. Cell numbers after 4 days were different in all three organisms. Sea urchin membranes had similar numbers to bovine collagen substrate and plastic controls, both sea star and sea cucumber membranes showed less. Further testing is needed to determine if this is a transient and temporary cell behavior or if it’s due to a toxic effect.

Overall sea urchins and partially sea stars display a major advantage when compared to bovine derived collagen. Further in vitro and in vivo studies are necessary to more deeply evaluate EDCM exploitability, including permeability, biodegradability and immunogenicity, all of these being key features to validate new biomaterials for human clinical applications.

Ferrario, C., Leggio, L., Leone, R., Di Benedetto, C., Guidetti, L., Cocce, V., . . . Sugni, M. (2016). Marine-derived collagen biomaterials from echinoderm connective tissues. Marine Environmental Research, In Press, In Press. doi:10.1016/j.marenvres.2016.03.007

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