Spontaneous Orthogonal Alignment of Smooth Muscle Cells and Endothelial Cells Captures Native Blood Vessel Morphology in Tissue-Engineered Vascular Grafts.

Tissue-engineered vascular grafts (TEVGs) have emerged as a potential alternative to autologous grafts for replacing small-diameter blood vessels during bypass surgery. The axial alignment of endothelial cells (ECs) and the circumferential alignment of smooth muscle cells (SMCs) are crucial for functional native blood vessels (NBVs). However, achieving this cellular alignment in TEVGs remains a formidable challenge. In this study, TEVGs were developed using a low-cost technique that aligned ECs axially and SMCs circumferentially within hours. The TEVGs comprised an electrospun polycaprolactone (PCL) layer and a gelatin methacryloyl (GelMA) cast layer. A freezing-induced alignment technique was developed that partially aligns the electrospun fibers axially, thereby promoting rapid axial alignment of ECs. Furthermore, SMCs cultured in a GelMA layer with intermediate stiffness (5-12 kPa) surrounding a PCL tube could promote conformation of the SMCs to the curvature of the PCL tube, resulting in their spontaneous circumferential alignment. Additionally, the TEVGs demonstrated mechanical properties similar to those of NBVs, which could facilitate future translation. This approach represents a significant advance in tissue engineering, enabling the fabrication of TEVGs with appropriate mechanical properties that recapitulate key NBV cell structural features within hours using a scalable and accessible method.

Peroxidase-catalyzed microextrusion bioprinting of cell-laden hydrogel constructs in vaporized ppm-level hydrogen peroxide.

Hydrogels were prepared by contacting air containing 10-50 ppm H2O2 with an aqueous solution containing polymer(s) possessing phenolic hydroxyl (Ph) moieties (polymer-Ph) and horseradish peroxidase (HRP). In this system, HRP catalyzes cross-linking of the Ph moieties by consuming H2O2 diffused from the air. The hydrogelation rate and mechanical properties of the resultant hydrogels can be tuned by controlling the H2O2 concentration in air, the exposure time of the air containing H2O2 to the solution containing polymer-Phs and HRP, and the HRP concentration. The shortest hydrogelation time of the solution stirred in air containing 16 ppm H2O2 was 6 s. Based on these findings, this hydrogelation system was applied to microextrusion bioprinting, in which bioink containing polymer-Phs, HRP, and cells were extruded into air containing H2O2. The superior cytocompatibility of the bioprinting method was confirmed by more than 90% viability, migration, and the spreading of mouse fibroblast 10T1/2 cells enclosed in the bioprinted hydrogels composed of derivatives of hyaluronic acid and gelatin, both possessing Ph moieties. These results demonstrate the great potency of HRP-catalyzed hydrogelation consuming H2O2 supplied in surrounding air for various biomedical applications, especially bioprinting.