Light-induced Cell Sheet Technology And Its Application In Tissue Engineering

Defects of maxillofacial caused by trauma or tumor often involve hard and soft tissues, some biological scaffolds have been successfully used for realizing the reconstruction of bone defect. However, to the limits of pre-vascularization by traditional biological tissues, there are usually poor activities in early bone building, deferring in bone reconstruction and degradation of the biological scaffolds, so pre-vascularization in the construction of bone tisstues becomes one of the most important things in the bone repairing; And by far the complex tissue repaired technologies are not mature enough, so more excellent tissue engineering technology has become a difficulty and hot issue in nowadays.In this study, we have developed a light-induced cell sheet technology based on the TiO2nanodot films, and took a introduction of its application in tissue engineering briefly. The main results are as follows:A spin-coating process and phase-separation-induced self-assembly method were used to prepare a pre-TiO2nanodot film, which could be transformed to an anatase TiO2dot film by500℃heat-treatment, and to an amorphous TiO2dot film by a hydrothermal treatment. There are more surface hydroxyl groups on the amorphous TiO2dot film, which leading several different characteristics between them. First, the adsorption status of BSA was multilayer on anatase TiO2nanodots, while presented monolayer type on amorphous TiO2nanodots, which further affected the conformation of attached fibronectin and subsequently resulted in better cell adhesion, proliferation and mineralization on anatase TiO2nanodots; And anatase TiO2nanodot film showed more surface hydroxyl groups or wettability shift than amorphous one after UVA illumination.The light-induced protein detachment is mainly due to the changes in the adsorption conformation of surface-bound protein on anatase TiO2nanodots after UV illumination. After UV365illuminated, the hydroxyl groups on the surface of TiO2nanodot were increasing, which would enhance the attraction to-NH3+groups of surface-bound protein, and decrease the exposed-NH3+groups on outer surface, making the outer surface of surface-bound protein change to less positively charged or even negatively charged. Subsequently, when the electrostatic repulsion between surface-bound proteins and outer-layer proteins in multilayer type adsorption was higher than the attraction of-NH3+and-COO-, the outer-layer proteins would desorb until to the saturation of surface hydroxyl groups on TiO2nanodot film.When using UV365nm with an intensity of2.0mW/cm2to illuminate the cell attached anatase TiO2nanodots for20mins under MEM, the detachment rate could surpass90%or an intact cell sheet could be acquired, while it was also compatible to other type of cells. The safety and viability of the light-induced cell harvest from TiO2nanodot film were proved by flow cytometry, DNA damage assessed, cells re-attachment, live-dead staining and surface hydroxyl groups assayed. And the light-induced cell sheet still contained an intact extracellular matrix, which could realize a faster defect repairing by the high efficiency of cell attaching.Considering the initial promoting role to osteoblast-liked cells by TiO2nanodot film and the high-usage of cell sheet, cell sheets obtained by light-induced technology had good repairing results in promoting osteogenesis and skin regeneration, which could realize the repairing of complex tissue defects in future.This study initiatively used light-induced cell sheet technology to obtain hMSCs cell sheets, and then constructed tissue engineered bone layer-by-layer assembly with sandwiched HUVECs in different orders to form three dimension reticular formation of capillary vessel in it finally, which would improve the poor activities in early bone building, deferring in bone reconstruction and degradation of the biological scaffolds caused by the limits of pre-vascularization in traditional biological tissues.This research demonstrated a light-induced cell or protein desorption phenomenon, clearly clarified the mechanism of the effect, optimized the condition of the technology, certified its safety and viability; At the same time, it provided a substantial advance for developing a complex cell sheets, which would further develop the formation of blood vessels in vitro and contribute to regenerative medicine in future.