The central region of the hypermineralized dolphin's bulla (ear bone), contains >95 wt% mineral with~10 wt% structurally incorporated carbonate. The mineral-bioapatite undergoes phase changes during heating. However, the step-by-step changes with increasing temperature are still unclear. Raman spectroscopy is proved to be a powerful technique in analyzing crystallographic transformation of bioapatite. The bulla was heated stepwise from 100°C to 900°C for the Raman analyses. Both positions and widths of Raman peaks were studied. Crystals of the bioapatite are stable up to 200°C. From 300°C to 600°C, the decrystallization is a gradual process with delicate changes rather than a sharp transformation. Carbonate is depleted at~500°C, and then decrystallization is terminated. At temperature over 600°C, bioapatite re-crystallizes and is transformed to OHAp at 700°C. The crystallinity is then gradually improved while the temperature increased to 900°C. No phases of β-tricalcium phosphate or calcium pyrophosphate were detected in this study.

Objectives To develop a rapid and simple method to fabricate intact, robust cell sheets from common cell culture dishes by combination of a macromolecular crowding (MMC) reagent and vitamin C.Results It was found that 3T3 broblasts or human bone marrow mesenchymal stem cells (hBMSCs) and their secreted cell derived extracellular matrices could be easily detached as intact cell sheets under gently pipetting after treated by MMC and vitamin C for 4 days. This method also allowed fabrication of functional multi-layered hepatic cell sheets by culturing 10×104 cells/cm2 HepG2 cells on top of con uent 3T3 broblast layers. What's more, MMC induced hBMSC cell sheets demonstrated 1.4 times larger area and 1.6 times greater cell number than that of cell sheets harvested from temperatureresponsive cell culture dishes.Conclusion MMC based method make it possible to fabricate various types of cell sheets more conveniently, economically, and thus may facilitate wide application of cell sheet technology.

Objectives To develop a rapid and simple method to fabricate intact, robust cell sheets from common cell culture dishes by combination of a macromolecular crowding (MMC) reagent and vitamin C. Results It was found that 3T3 fibroblasts or human bone marrow mesenchymal stem cells (hBMSCs) and their secreted cell derived extracellular matrices could be easily detached as intact cell sheets under gently pipetting after treated by MMC and vitamin C for 4 days. This method also allowed fabrication of functional multi-layered hepatic cell sheets by culturing 10×104 cells/cm2 HepG2 cells on top of confluent 3T3 fibroblast layers. What’s more, MMC induced hBMSC cell sheets demonstrated 1.4 times larger area and 1.6 times greater cell number than that of cell sheets harvested from temperature-responsive cell culture dishes. Conclusion MMC based method make it possible to fabricate various types of cell sheets more conveniently, economically, and thus may facilitate wide application of cell sheet technology.