Two-photon polymerization of poly(caprolactone) and extracellular matrix polymer scaffolds for retinal tissue engineering

Arwin Shrestha

doi:10.17077/etd.005722

Central to retinal degeneration is the loss of photoreceptor cells critical for vision. The regeneration of these photoreceptors is comprised of several complex pathways. Thus, cell replacement therapies are often considered instead. Injection of the appropriate cell type is not sufficient for replacement, rather a tissue engineered retinal graft is necessary. Two-photon polymerization (TPP) was utilized for scaffold fabrication to create a graft design. TPP grants the ability to create microstructures theoretically capable of supporting cells for transplantation into the subretinal layer. In this work, multiple polymer formulations, both synthetic and biological, were successfully tested using this polymerization method to create these structures. The synthetic polymer, poly(caprolactone) (PCL), had previously displayed biocompatible results and scaffold fabrication capabilities. Here, the scaffold design was improved upon to allow cell-to-cell and cell-to-tissue interaction by varying the printing parameters, slicing and hatching, through an iterative design process. Conversely, biopolymers derived from the extracellular matrix (ECM), collagen, gelatin, and hyaluronic acid (HA), required initial experimentation to determine the additional printing parameters, scanning speed and percent laser power, required for polymerization. The photoinitiator, an ingredient required for photopolymerization, used with the biopolymers differed from PCL so different printing parameters were expected for polymerization to occur. In addition to TPP of the biopolymers, ultraviolet (UV) polymerization experiments were conducted to examine their crosslinking capabilities. Both UV and TPP structures were tested for biocompatibility with induced pluripotent stem cells (iPSCs) and retinal progenitor cells (RPCs) and were not considered cytotoxic. In addition to testing the biopolymers separately, a 50/50 mixture of gelatin and HA was evaluated. Alone, HA was difficult to create structures with TPP, however when combined with gelatin, scaffold fabrication was feasible. Furthermore, when analyzing the biocompatibility of the TPP scaffolds with RPCs, the 50/50 gelatin/HA scaffolds displayed increased neuronal development than the homogenous gelatin scaffolds. Overall, the gelatin/HA composition resulted in the most favorable outcomes for scaffold fabrication and biocompatibility. The purpose of experimenting with the biopolymers was to identify ECM molecules capable of crosslinking for implementation in retinal disease modeling and therapeutic testing. In addition to polymer testing with TPP, several devices were created with a dual extrusion three-dimensional (3D) printer. These devices were designed to increase efficiency and resolve complications encountered during experimentation. One such complication is the lack of consistency in the wound healing or scratch assay. Traditional use of pipette tips encourages user error and variable scratches. Conversely, the 3D printed model, Track Scratcher explained within Chapter 4, eliminates these inconsistencies. Another complication is the loading of cells onto UV polymerized PCL films which have, customarily, been attempted via passive transport. However, this transport method was largely unsuccessful, so a cell loading device was created as an active transport apparatus. Both of these devices were successful in increasing efficiency and accomplishing their designed purpose. In summary, the ability to create 3D structures enhances the capabilities of improving on current techniques, whether for laboratory experiments or for tissue engineering.

Two-photon polymerization of poly(caprolactone) and extracellular matrix polymer scaffolds for retinal tissue engineering

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