Tissue engineering aims to replace missing or damaged tissues and restore their functions. Three-dimensional (3D) printing has been gaining more attention because it enables the researchers to design and produce cell loaded constructs with predetermined shapes, sizes, and interior architecture. In the present study, a 3D bioprinted corneal stroma equivalent was designed to substitute for the native tissue. Reproducible outer and inner organization of the stroma was obtained by optimizing printing conditions such as the nozzle speed in the x–y direction and the spindle speed. 3D printed GelMA hydrogels were highly stable in PBS during three weeks of incubation (8% weight loss). Live–Dead cell viability assay showed 98% cell viability on day 21 indicating that printing conditions were suitable for keratocyte printing. Mechanical properties of the cell loaded 3D printed hydrogels increased 2-fold during this incubation period and approached those of the native cornea (ca. 20 kPa vs. 27 kPa, respectively). Expression of collagens types I and V, and proteoglycan (decorin) in keratocytes indicates maintenance of the phenotype in the hydrogels. Transparency of cell-loaded and cell-free hydrogels was over 80% (at 700 nm) during the three week culture period and comparable to that of the native cornea (85%) at the same wavelength. Thus, GelMA hydrogels bioprinted with keratocytes mimic the biological and physical properties of the corneal stroma with their excellent transparency, adequate mechanical strength, and high cell viability.

Methacrylated gelatin (GelMA) hydrogels were prepared to serve as corneal stroma equivalents. They were highly transparent (ca. 95% at 700 nm), mechanically strong and withstood handling and had high human corneal keratocyte viability (98%) after 21 days of culture period. In order to test the in vivo performance of the cell free GelMA hydrogels a pilot in vivo study was carried out using eyes of two white New Zealand rabbits. Hydrogel was implanted in a mid-stromal pocket created and without suture fixation, and observed for 8 weeks under a slit lamp. No edema, ulcer formation, inflammation or infection was observed in both the control (sham) and hydrogel implanted corneas. Corneal vascularization on week 3 was treated with one dose of anti-VEGF application. Hematoxylin and Eosin staining showed that the hydrogel was integrated with the host tissue with only a minimal foreign body reaction. Results demonstrated some degradation in the construct within 8 weeks as evidenced by the decrease of the diameter of the hydrogel from 4 mm to 2.6 mm. High transparency, adequate mechanical strength, biocompatibility, and well integration with the host tissue, indicates that this hydrogel is a viable alternative to the current methods for the treatment of corneal blindness and deserves testing on larger number of rabbits and more extensively using microscopy, histology and immune histochemistry.

Here, we describe the characterization of tooth-germ organoids, three-dimensional (3D) constructs cultured in vitro with the potential to develop into living teeth. To date, the methods used to successfully create tooth organoids capable of forming functional teeth have been quite limited. Recently, hydrogel microparticles (HMP) have demonstrated utility in tissue repair and regeneration based on their useful characteristics, including their scaffolding ability, effective cell and drug delivery, their ability to mimic the natural tissue extracellular matrix, and their injectability. These outstanding properties led us to investigate the utility of using HMPs (average diameter: 158 ± 32 µm) derived from methacrylated gelatin (GelMA) (degree of substitution: 100%) to create tooth organoids. The tooth organoids were created by seeding human dental pulp stem cells (hDPSCs) and porcine dental epithelial cells (pDE) onto the HMPs, which provided an extensive surface area for the cells to effectively attach and proliferate. Interestingly, the cell-seeded HMPs cultured on low-attachment tissue culture plates with gentle rocking self-assembled into organoids, within which the cells maintained their viability and morphology throughout the incubation period. The self-assembled organoids reached a volume of ~50 mm³ within two weeks of the in vitro tissue culture. The co-cultured hDPSC-HMP and pDE-HMP structures effectively attached to each other without any externally applied forces. The presence of polarized, differentiated dental cells in these composite tooth-bud organoids demonstrated the potential of self-assembled dental cell HMPs to form tooth-bud organoid-like structures for potential applications in tooth regeneration strategies.