*3.2. Microscopy Fiber Analysis*

Fluorescence microscopy—Collagen fibers isolated from the coral were visualized under a fluorescence microscope at 305–450 nm (Optiphot, Nikon, Tokyo, Japan).

Phase contrast microscopy—Live cell cultures were observed under phase contrast microscopy and digital photography (Optiphot, Nikon, Tokyo, Japan).

Scanning electron microscopy (SEM)—For scanning electron microscopy, subsamples were fixed in 4% glutaraldehyde in filtered seawater (0.22 μm FSW), decalcified as described above, dehydrated through a graded series of ethanol up to 100%, and critical point dried with liquid CO2. The preparations were fractured under a compound microscope, using the tips of fine forceps, and the gastrovascular cavities were then carefully exposed. Next, they were gold-coated and examined under a scanning electron microscope (SEM Jeol-840a, Jeol LTD., Tokyo, Japan). In order to obtain fiber preparations, fibers were isolated as explained above and stored in 70% ethanol. They were then processed, coated with gold-palladium alloy, and examined at high vacuum under an environmental scanning electron microscope (SEM and ESEM, JSM-6700 Field Emission Scanning Electron Microscope, Jeol LTD., Tokyo, Japan). The diameters of collagen fibers and fibrils were obtained from the images, using the ImageJ software.

#### *3.3. Bio-Composite Fabrication*

The extracted fiber bundles were sterilized (Figure 2A), arranged on a metal frame, and then inserted into a dialysis membrane (6000–8000 MWCO, Spectra Por, Spectrum Labs Inc., Rancho Dominguez, CA, USA) together with 3 mL sodium alginate solution (3% *w*/*v* in DDW (Protanal LF 10/60, FMC BioPolymer, Philadelphia, PA, USA)). The membrane was sealed, flattened, and soaked in CaCl2 (0.02 M Ca for Tissue culture (TC) experiments or 0.1 M for mechanical testing) solution used as a cross-linker through diffusion for 48 h at room temperature to allow the gelation of the alginate to hydrogel. The bio-composite was then removed from the membrane and the frame was sterilized by immersion in 70% ethanol and further used for cell seeding. Alginate hydrogel samples were fabricated by the same protocol, excluding the embedding of fibers.
