Our group is interested in tissue engineering for urethral reconstruction performed in patients with urethral strictures or congenital disorders. The CS is an integral part of the urethra and important in supporting the function of the urethra. In case of a healthy CS, success rates of replacement with epithelial tissue like skin or oral mucosa can reach up to 90%. However, in case of severe fibrosis or absence of CS in congenital disease (hypospadias) success rates are lower. Currently there is a lack of tissue-engineered solutions for replacement/ regeneration of urological tissues, like ureters and the urethra/CS. Such tissues present a complex tubular organization with different cell layers. Given the important role of the CS in urethra function, tissue engineering of the urethra should be combined with reconstruction of the CS [1]. We showed that the CS is a three-layered, highly vascularized structure with distinct distribution of extracellular matrix (ECM) components (Figure 1A). Therefore, we hypothesize that by an innovative casting approach to build multilayered tubular constructs based on micro-fiber reinforced hydrogels CS tissue constructs can be generated that mimic the structure/organization of the native tissue.

A mold with three chambers, representing the three layers of the CS (Fig. 1B), was designed, and fabricated using polydimethylsiloxane (PDMS) molding. The chambers were loaded with gelatin-transglutaminase hydrogels (mTG gel) containing a coculture of endothelial cells and pericytes (layer 1 and 3) and smooth muscle cells (SMCs, layer 2). 
A melt-electrospun poly(caprolactone) (PCL) fibre mesh was incorporated at the base of the construct to serve as a porous support for the hydrogels and to roll the construct into a multilayered tubular construct (Fig. 1C). The hydrogels were mechanically tested and compared to native tissue (equine urethra).

The custom-made mold was successfully designed and made from PDMS. The PDMS mold was easy in terms of fabrication and handling and ensured excellent mold removal from the hydrogel. The mTG gel, containing either a combination of HUVECs and pericytes in chamber 1 and 3 or SMCs in chamber 2, can be successfully casted and rolled (Fig. 2A-B). The encapsulated cells were cultured up to 2 weeks and showed good cell viability and functionality. Within two weeks little capillary-like structures were formed in layer 1 and 3 (Fig. 2C) and the SMCs express elastin (Fig. 2D). The compressive modulus of native tissue (equine urethra and CS) was similar to the mTG gel (Fig. 1B).

With this innovative gel casting approach it is possible to create multilayered tubular constructs: we were able to successfully roll the encapsulated gel into a tubular construct with distinct composition per layer. The rolling procedure did not influence the viability and functionality of the encapsulated cells. Cell survival up to two weeks has been achieved as well as functionality. The encapsulated cells are homogenously distributed throughout the gel and the HUVECs are able to form vascular networks, stabilized by pericytes, in the mTG hydrogels. Furthermore, SMCs show a high viability after encapsulation and largely express elastin which is required for mimicking the elastin-rich region of the corpus spongiosum. Mechanical testing has proven that the gel is similar to native tissue in terms of the compressive modulus.

This approach towards tissue engineering of multilayered tubular structures may be applicable to the urological field (to help engineer ureters or urinary diversions), as well as in other fields of soft tissue engineering. In future studies, more research should be done in further standardization and optimization of the (fiber-reinforced) gels and fabrication of the electrospun meshes. This is required for mimicking the mechanical properties of native tissue as well as optimizing vascular network formation. Next steps will be up-scaling of our casting approach to achieve grafts of clinical relevant sizes and, in parallel, testing in laboratory animals whether the vascular networks produced in the hydrogel will adapt to the native vasculature.

de Kemp, V., et al. (2015). Tissue engineering for  human urethral reconstruction: systematic review ofrecent literature. PloS one 10, e0118653