Sponge-Mimic biomaterial Intercalated Silicate-Phosphate Tubules (SMTS) for 3D Cell Culture and Zinc incorporated Biomimetic Sponge Battery Materials

Hydrothermal vent systems at the interface with ocean waters form intricate mineral structures.[1] These structures can vary in size from micrometers to several meters in height. Hydrothermal vents are considered as the chemical foundries in which the building blocks of life was forged: (it has been shown minerals present in these systems can catalyze the synthesis of the small biomolecules of life and their polymerization to produce DNA and proteins)[2] ; bacteria have been observed colonizing these structures. The oldest Fossils (4.3 Billion year old) hitherto discovered are micrometer sized iron rich (hematite) tubules from ancient hydrothermal vents associated with microbes.[3] Chemical gardens serve as an excellent model system to mimic hydrothermal vents. They are produced by seeding or injecting water soluble salts of several multivalent cations into a highly concentrated solution of sodium silicate or phosphate. The seeded metal salt, for example, calcium chloride, starts dissolving in the aqueous medium, this triggers the formation of a semipermeable membrane of calcium silicate; since the ionic concentration of Ca2+ is higher inside the membrane, osmotic pressure increases causing the top of the membrane to rupture, the membrane formation and rupture process repeats resulting in the formation of tubular mineral assemblies.[4] This emerging discipline of producing self-assembled constructs of tubular semipermeable membranes composed of inorganic amorphous and crystalline phases has been recently defined as chemobrionics.[5]
The scaffolds of sea sponges are composed of an intercalating network of the protein spongin with calcium silicate/carbonate on which choanocytes (sponge cells) are organized and held in position by cell adhesion proteoglycans, forming the multicellular organism.[6,7] Sponges are considered the earliest form of multicellular animal life (Figure 1(1)). The scaffold of tubular sponges is analogous in construction to hydrothermal vent fields and chemical gardens with a marked difference: the protein component is missing in the latter constructs, which are purely mineral structures. We have recently achieved sponge biomimicry and redefined the chemical garden experiment by seeding a concentrated sodium silicate-potassium phosphate solution containing solubilized gelatin with calcium chloride to produce protein intercalated silicate-phosphate tubules-Sponge mimetic Tubules (SMTS) which are essentially bio-hybrid chemical gardens (Figure 1(2 and 3)).[8,9] The final constructs bear a remarkable morphological resemblance to the scaffolds of tubular sponges. This discovery transforms the classic chemical garden experiment into a highly innovative technology to create advanced bio inspired materials for a range of potential applications including 3D cell culture (bioengineering, origin of life research) and battery material development.
Figure 1. Figure caption. 1) Tubular Sponge. 2) Chemical garden based sponge mimic tubule (SMT): protein intercalated tubular scaffolds constructed by seeding CaCl2 in a solution composed of 3M Sodium silicate, 0.5M K2HPO4 and 15% solubilized gelatin at 60oC. 3) Scanning Electron Micrograph of gelatin-intercalated silicate-phosphate scaffold tubes, Scale bar 200µm. a) Marine Dinoflagellate strain Pyrocystis lanula cultured on the surface of 15% gelatin scaffold (SMT) modified prior to cell culture with cell adhesion molecules, imaged via confocal laser scanning microscopy (CLSM); the live dinoflagellate chloroplasts fluoresce in the far red region. (b) Calcium chloride seeded 15% gelatin-silicate-phosphate tubular scaffold (SMT) coated with cell adhesion molecule chitosan on which H9C2 cells (rat cardiomyocytes) are cultured; the cells are labeled with the cytoplasmic live cell indicator fluorescein diacetate and viewed using a Zeiss live cell imager: the entire construct is covered with live H9C2 Cells which fluoresce green. Scale bars (a) 200 µm, (b) 10 µm. (c) Honeycomb sponge (d) Scanning Electron Micrograph of Zinc powder incorporated chemical garden sponge biomaterial: constructed by mixing CaCl2 into a solution composed of 3M Sodium silicate, 0.5M K2HPO4, 15% solubilized gelatin and 10% suspended Zinc powder at 60oC; the potential battery material is spongy with numerous pores. Scale bar 100 µm.

Specific aim 1: To recreate a biomimetic sponge model to validate a new theory we propose here for the origin of ocean sponges.
Specific Aim 2: Produce and program the surface chemistry of SMTs for mammalian cell adhesion/differentiation and optimize conditions for mammalian 3D cell culture applications including bioengineering.
Specific aim 3: To create nano/micro-particle incorporated sponge mimetic materials for battery applications.

Specific Aim One: To recreate a biomimetic sponge model to validate a new theory we propose here for the origin of ocean sponges.












































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