Scaffold-free tissue engineeing with surface-functionalized thermally expandable hydrogels

Abstract

Tissue engineering has been developed by combination of biology, medicine, and material science to restore or improve functions of damaged tissue and provide ex vivo tissue model for exploit of biological phenomenon. Functional artificial tissue can be acquired by mimicking structural features of target tissue possessing various scales from micro to multi-layered structure and cellular microenvironment including extracellular matrix (ECM) in both of two and three dimensional (3D) aspect. Various methodology such as bioprinting have been developed, and modulation of biological functions by using bioactive molecule also have been extensively studied. However, shortcomings involving inflammatory reaction, acidic byproducts and undesired residue of polymer substrate still not have been clearly solved. Meanwhile, development of stimuli-responsive materials alternating cell binding affinity depending on external signal enabled separation of engineered tissue from culture substrate. This acquisition method referred as scaffold-free harvest technique has been studied since it can bypass the aforementioned problems. Additional advantage of preservation of intact cell-cell and cell-ECM was capitalized on clinical purpose and formation of ex vivo artificial 3D tissue possessing improved functional similarity with natural tissues. Despite of previous accomplishments, conventional scaffold-free harvest techniques have faced challenges of laborious process and especially recapitulation of natural tissue structures with multiple scales. Herein, I developed surface modified thermally expandable hydrogel for harvest and delivery of microtissues with various scales from spheroid to multi-layered assembly. Thermally expandable hydrogel composed of Tetronic® with crosslinkable end group was prepared to generate cell detaching force in response to temperature decrease. On the substrate, I conducted surface modification mediated by mussel-inspired polydopamine (PD) molecule. Exquisite control of cell adhesion property via parameters of reaction time, pH enabled sheet formation with multiple cell types and robust detachment of the sheets with high efficiency and viability with rapid process time (10 min). Easy applicability of PD on microfabrication techniques enabled simple patterning of PD on the hydrogel surface with micrometer scaled structure. Microtissues with desired form such as fiber and spheroid were harvested by using the patterned substrate, and potential availability on cell based therapy and 3D ex vivo tissue culture model were proved by delivering microtissues to in vitro and in vivo targets. Secondary coating of cell adhesive molecule on PD coated surface was able to support formation of multi-layered assembly. Delivered multi-layered from the substrate presented not only preserved ECM structure but also release of growth factor in vitro which might result of inducing vessel ingrowth after transplantation on mouse subcutaneous model. Collectively, cell interactive surface was developed on Tetronic®-based thermally expandable hydrogel by utilizing various applicability of PD, and facilitated harvest of microtissues recapitulating various scaled of natural tissues. The developed system can be widely utilized for tissue engineering and regenerative medicine.