Dynamic echinoderm collagenous tissue: inspiration for biomimetic substrates

Introduction Biomimetic scaffolds consisting of various materials (eg. collagen, chitosan) have been developed recently1. Alternative routes of scaffold engineering are required. Echinoderms (starfish, sea urchin, etc.) possess unique connective tissues (Mutable Collagenous Tissues: MCTs), that undergo rapid and drastic changes in their mechanical properties2. MCTs are composed of collagen fibrils comparable to those of mammals. Considering this, MCTs could be an inspiration for new materials capable of changing their molecular and structural conformation in response to external stimuli. This contribution presents a review of the biological features of these dynamic connective tissues in the context of developing MCT-inspired matrices offering an excellent environment for tissue regeneration as well as for applications requiring plasticization/stiffening of connective tissues. The first employment of these new scaffolds as cell culture substrata is presented in echinoderm models. Materials and Methods In order to obtain MCTs in a compliant or stiffened state, sea urchins (Paracentrotus lividus) were treated with propylene phenoxetol or with acetylcholine respectively. Typical MCTs, i.e. compass depressor ligaments (CDLs) and peristomial membranes (PMs), were prepared for light (LM) & electron microscopy (TEM, FEG-ESEM) through fixation in glutaraldehyde 2%, post-fixation in OsO4 1% and uranyl acetate 2%, dehydration with ethanol series, embedding in Epon Araldite 812. For FEG-ESEM, uranyl acetate step and resin embedding were excluded and samples were dried with HMDS before the observation. Collagen fibrils were extracted stirring PMs in a disaggregating solution (0.1 M Tris-HCl pH8, 0.5 M NaCl, 0.05 M EDTA, 0.2 M β-mercaptoethanol) and stained for TEM analysis with phosphotungstic acid. Extracts were stirred in 4 M guanidine-HCl in 0.05 M sodium acetate (pH 5.8) or 0.1 N NaOH in order to remove PGs (proteoglycans) and cells. Intact peristomial membranes were treated with the same solutions, paraffin embedded, sectioned and stained (Milligan’s Trichrome and Alcian Blue) in order to check the efficiency of cell and PG removal. Cuprolinic Blue (CB) staining of GAGs (glycosaminoglycans) was performed on PMs, CDLs and extracted fibrils. Collagen and GAG quantification was performed with Sirius red3 and Alcian blue respectively. Sea urchin coelomocytes were cultured on sea urchin PMs decellularized with, 0.1% EDTA in 10mM TRIS and 0.1% SDS in 10mM TRIS4. Cell behavior was checked through LM, TEM and confocal microscopy. Results and Discussion TEM images showed that collagen fibril orientation was parallel in stiffened CDLs and irregularly distributed in compliant CDLs. Interfibrillar distance was reduced in stiffened samples suggesting that, in order to maintain a stiffened condition, fibrils need to be side by side so that stabilizing interfibrillar bridges could form. CB revealed that PGs are regularly associated with collagen fibrils. GAG quantification was similar in different mechanical states, thus suggesting that differences could be related to GAG types present. Alcian blue staining was not affected by guanidine chloride and strongly reduced by NaOH. Since guanidine chloride removes non-covalently bound PGs from collagen fibrils, and it had no effect on PM, covalently bound PGs dominate in this tissue. Presence of collagen fibrils in extracts was confimed by TEM and Sirius red. Coelomocytes cultured on decellularized PMs adhered and produced interconnecting filopodia. References 1. Gelain F., Horii A., Zhang S. (2007). Macromol Biosci, 7: 544-551. 2. Wilkie I.C. (2005). In: Matranga V. (eds). Progress in Molecular and Subcellular Biology, Subseries Marine Molecular Biotechnology, Echinodermata, Springer-Verlag, Berlin Heidelberg, pp:167-199. 3. Taşkiran D., Taşkiran E., Yercan Y., Kutay F.Z. (1999) Analytical Biochemistry, 150: 86-90. 4. Ott H.C., Matthiesen T.S., Goh S., Black L.D., Kren S.M., Netoff T.I., Taylor D.A. (2008). Nature Medicine, 14 (2): 213-221. Acknowledgements The present work was financed by the CARIPLO Foundation (MIMESIS Project)