3D Chitin Scaffolds of Marine Demosponge Origin for Biomimetic Mollusk Hemolymph-Associated Biomineralization Ex-Vivo

Structure-based tissue engineering requires large-scale 3D cell/tissue manufacture technologies, to produce biologically active scaffolds. Special attention is currently paid to naturally pre-designed scaffolds found in skeletons of marine sponges, which represent a renewable resource of biomaterials. Here, an innovative approach to the production of mineralized scaffolds of natural origin is proposed. For the first time, a method to obtain calcium carbonate deposition ex vivo, using living mollusks hemolymph and a marine-sponge-derived template, is specifically described. For this purpose, the marine sponge Aplysin aarcheri and the terrestrial snail Cornu aspersum were selected as appropriate 3D chitinous scaffold and as hemolymph donor, respectively. The formation of calcium-based phase on the surface of chitinous matrix after its immersion into hemolymph was confirmed by Alizarin Red staining. A direct role of mollusks hemocytes is proposed in the creation of fine-tuned microenvironment necessary for calcification ex vivo. The X-ray diffraction pattern of the sample showed a high CaCO3 amorphous content. Raman spectroscopy evidenced also a crystalline component, with spectra corresponding to biogenic calcite. This study resulted in the development of a new biomimetic product based on ex vivo synthetized ACC and calcite tightly bound to the surface of 3D sponge chitin structure. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Naturalis Biodiversity Center Ministerio de Ciencia, Tecnología y Medio Ambiente, CITMA: 18-54-34007 Deutsche Forschungsgemeinschaft, DFG: HE 394/3 Russian Foundation for Basic Research, RFBR Russian Science Foundation, RSF: 18-13-00220 57397326 Ministerstwo Nauki i Szkolnictwa Wyższego, MNiSW: 03/32/SBAD/0906 PPN/BEK/2018/1/00071 Deutscher Akademischer Austauschdienst, DAAD Sächsisches Staatsministerium für Wissenschaft und Kunst, SMWK: 02010311 Funding: This work was partially supported by the DFG Project HE 394/3, SMWK Project no. 02010311 (Germany), as well as DAAD-Italy Project “Marine Sponges as Sources for Bioinspired Materials Science” (No. 57397326). The sampling conducted in Cuba (V.I.) and Curaçao (V.I. and B.H.) was funded by RFBR and CITMA, according to the research project 18-54-34007 (grant), and Temminck-Fellowships from the Naturalis Biodiversity Center, respectively. M.W is financially supported by the Polish National Agency for Academic Exchange (PPN/BEK/2018/1/00071). Tomasz Machałowski is supported by DAAD (Personal ref. no. 91734605). Yuliya Khrunyk is supported by the Russian Science Foundation (grant no. 18-13-00220). This research was partially funded by the Ministry of Science and Higher Education (Poland), as financial subsidy to PUT No. 03/32/SBAD/0906.