BACKGROUND: Historically, tissue engineered urethral scaffolds consisted of a monolayered construct seeded with one or more cell lines. However, the anterior urethra is a multilayered structure with dedicated cell lines residing in each of these layers. Monolayered scaffolds fail to recapitulate the distinct microenvironmental cues necessary to spatially direct the growth of these dedicated cell lines. Mechanical cues determined by tensile properties are a key element to direct cell growth and physiology. In our previous work, highly elastic GelMA/ELP monolayer scaffolds with tensile properties similar to the whole native urethra were developed by electrospinning. The aim of this study was to modify these materials to design two distinct bioinks with hypothesized target tensile properties for UC and SMC growth, respectively. The bioinks were used to 3D print a hollow bilayer scaffold that retains its structural integrity.
METHODS: Bioinks were synthesized to achieve target mechanical properties for the inner and outer layer. Gelatin-methacryloyl (GelMA) was used for the inner layer due to its cell binding sites and soft mechanical properties. To increase the extensibility of the outer layer, the second bioink adds methacrylated elastin-like polypeptide (mELP) to GelMA. The bioink candidates with varied concentrations were characterized by NMR spectroscopy, rheology, tensile testing, swelling, and degradation assays. The optimized bioinks selected based on the materials data were used to print a bilayer structure using the FRESH printing technique inside a 1.5 w/v% Carbopol bath. Urothelial, smooth muscle, and fibroblast cells are currently being encapsulated inside the bioinks. Data was described using mean and SEM; a two-way ANOVA evaluated between-group differences.
RESULTS: Varying the GelMA/mELP ratio has a substantial effect on the mechanical properties of the bioink candidates (Fig A-E). The addition of mELP to GelMA decreased ultimate tensile strength (UTS) (A) and increased extensibility (B). The GelMA/mELP bioinks showed enhanced degradation, aligning with anticipated cell proliferation and extracellular matrix deposition (E). A 10 w/v% GelMA bioink provides the required softness of the inner layer. The 1:1 GelMA/mELP ratio was selected for future SMC encapsulation because it provides sufficient structural support (high UTS) while also showing high extensibility. Excellent cell viability was observed through day 5 for the fibroblasts encapsulated in these bioinks (F). A bilayer hollow tube was successfully 3D printed that retained its 3D structure after removal from the FRESH bath (G).
CONCLUSIONS: Two bioinks based on GelMA and GelMA/mELP were developed to provide the microenvironmental cues for UC growth and SMC growth, respectively. A bilayer hollow tissue construct with the bioink targeting UC growth as the inner layer and the one targeting SMC growth as the outer layer was successfully 3D printed. To verify whether target properties are achieved, UC and SMC are currently being encapsulated inside the bioinks. Future incorporation of nanoparticles or bioactive factors are planned to enhance vascularization and regeneration. In addition, personalized urethral tissue constructs will be 3D printed.
