Professor Sungkyunkwan University (SKKU) Suwon, United States
Introduction:: In traditional tissue engineering field, bulk-type hydrogels made from natural source, such as gelatin, collagen, chitosan, and hyaluronic acid, or synthetic polymer, such as poly(vinyl alcohol), poly(ethylene glycol), and polyurethane, have been used to re-construct damaged tissue. However, bulk hydrogels lack microporosity for cell infiltration or vascularization near target sites, resulting in delayed tissue regeneration until their degradation. As tissue regeneration has complex interactions with surrounding tissues and cells, recently, the granular hydrogels formed by aggregating particles have been studied for wound repair. The interparticle-porosity between packed particles in granular hydrogels is beneficial for diffusion of nutrients and the distribution of cells. In this study, we proposed a novel granular hydrogel made of microbeads based on decellularized tilapia skin to construct 3D scaffold in the wound site. The ECM-based granular hydrogels promoted the recruitment of cells around the wound area, resulting in rapid wound regeneration and tissue remodeling in vivo, suggesting that tilapia skin ECM-based granular hydrogel could serve as a promising biomaterial platform for tissue regeneration.
Materials and Methods:: The tilapia-derived (decellularized ECM, dECM) granular hydrogel was fabricated by mixing microbeads from decellularized tilapia skin and hyaluronic acid catechol (HA-catechol) as adhesive between microbeads. For preparing microbeads, first, tilapia skins were obtained from a nearby fish farm, washed with acetone and DI water, and decellularized with 0.1 % Triton X-100 as detergent to remove cell components in tilapia skin. The decellularized tilapia skins were solubilized with 0.01M HCl with pepsin for 48 hr at RT. Next, a flow-focusing microfluidics device was prepared in which tilapia skin solution (dECM) was injected as dispersed phase and mineral oil (with surfactant) was injected as continuous phase. The pre-made dECM microdroplet passed through the tubing, met high pH oil supplemented with propylamine, and physically cross-linked to form dECM microbeads. For dECM granular hydrogels, dECM microbeads and HA-catechol are mixed in a syringe at a certain volume ratio. To investigate whether it can be used as a 3D scaffold, L929 cells were cultured in dECM granular hydrogels and dECM bulkgels. Additionally, to determine its effect on wound healing, a full thickness wound mouse model was created and dECM granular hydrogels were injected using 22G needle, followed by observation for 14 days.
Results, Conclusions, and Discussions:: The decellularized tilapia skin was confirmed to have removed the nucleus of the cells through H&E staining. The amount of sulfated glycosaminoglycan and hydroxyproline, the major components of the ECM, were checked and the results showed that there was not a significant difference in their content after decellularization. In addition, it was quantitatively confirmed that the amount of DNA had decreased by more than 90%.
The size of fabricated dECM microbeads can be controlled depending on the flow rate of the dispersed phase and the continuous phase, and the channel size of the microfluidics chip. As a result, the size of dECM microbeads can be adjusted from approximately 70 to 120 µm in a microfluidics chip with a 40 µm channel size, and from approximately 120 to 240 µm in a microfluidics chip with an 80 µm channel size.
To confirm the effectiveness of 3D cell culture, L929 cells were encapsulated in dECM granular hydrogel and dECM bulkgel counterpart, and cultured for 7 days. The live/dead assay showed that the cells in the granular hydrogel had attached to the particle surface and proliferated, while those in the bulkgel were unable to proliferate due to lack of space and were dying off.
To confirm the wound healing effects, granular hydrogel and bulk were applied to a full thickness wound model and the wound size was monitored. On the fourth day, the granular hydrogel resulted in about 60% recovery while the bulk hydrogel showed 37% of tissue regeneration, demonstrating faster tissue regeneration compared to the bulkgel. Furthermore, after 14 days, H&E staining showed that both the dermis layer and hair follicles in the epidermis had fully regenerated in the granular hydrogel. In conclusion, we were able to produce dECM microbeads from non-mammalian fish for the first time and demonstrated their potential for use in tissue regeneration by creating a granular hydrogel incorporating HA-catechol. Our finding suggests that decellularization-based scaffolds can be useful materials for tissue regeneration and can be further developed with 3D printing devices for various applications.