(K-438) Altering the Stiffness of Two Photon Polymerized Human Lamina Cribrosa Models

Introduction:: Sub-track: Biomechanics of Engineered Tissues

Primary Open Angle Glaucoma (POAG) is one of the leading causes of irreversible blindness, estimated to affect 110 million people by 2040 [1]. During the progression of the disease, the lamina cribrosa (LC) becomes remodeled and undergoes cupping in patients’ optic nerve heads (ONH) [2]. The LC is a porous, collagenous disc located in the ONH which mechanically supports retinal ganglion cell (RGC) axons as they exit the eye (Figure 1) [3]. LC remodeling is associated with RGC death and which leads to blindness. Elevated intraocular pressure (IOP) can be a risk factor of POAG. It is suspected that elevated IOP may play a role in LC remodeling and cupping, however there are cases of POAG without elevated IOP [2].
Because the LC undergoes these mechanical changes and stress, it would be very beneficial to model the LC. Two photon (TP) polymerization may be used to produce high fidelity models that match the geometry of the LC. These models will allow researchers to understand the mechanics of each patient’s LC. The purpose of this study was to alter the stiffness of a fabricated LC and to investigate differences in anterior to posterior displacement.

Materials and Methods:: We produced model LCs using an Olympus BX51 upright laser-scanning microscope (Insight DS+, Spectra Physics, Santa Clara, CA) controlled with a custom Python script. The laser polymerized Nanoscribe™ IP-PDMS at a wavelength of 780nm (Figure 3).
We attempted to alter the stiffness by polymerizing models an additional time prior to development. Line average is typically used in imaging, where the laser scans a number of times before averaging the intensity of each pixel. By increasing the line average from 1 to 2, we allowed the laser to run two times in the same area.
We developed the models for 20 min in 70% ethanol before allowing them to air dry. We then placed them over a biopsy punched hole in a PDMS sheet. We sealed and cured the edges with liquid PDMS.
We placed the mounted LCs into a custom Intravital Bioreactor, and applied pneumatic pressure using an Elveflow™ OB1 Microfluidic Flow Controller (Figure 2) at 0, 5, 15, 30, and 45mmHg. Typical IOP ranges from 11mmHg to 21mmHg, and anything above 21mmHg is considered elevated IOP, which is a risk factor for glaucoma [4]. To prevent leakage through the pores, we placed a thin (~0.4mm) PDMS sheet on top of the models before sealing the chamber.
We used TP imaging to measure axial displacement. We tracked the z-depth of a single point towards the center of the model as it moved along the anterior posterior axis with increasing pressure. We recorded the relative change as displacement.

Results, Conclusions, and Discussions:: Results
Results are summarized in Figure 4. 1x Polymerized LCs had a lower average displacement than 2x Polymerized LCs at all pressures. We conducted two sample t-tests at each pressure, which indicated the differences between the displacements of each group are not significant.

Discussion
Preliminary data shows that an additional polymerization increases the displacement of an LC model, although this increase is not statistically significant. These results are unexpected, since an additional polymerization should further cure and crosslink the photoresin, resulting in a stiffer model and decreased displacement. It is possible that running the laser over the same area an additional time compromises the integrity of already polymerized material, resulting in a weaker structure and a larger displacement.
Future work should evaluate whether additional polymerizations continue this trend and result in larger displacements. We should test more samples to determine whether this difference, if any, is statistically significant. Additionally, we should investigate other methods of stiffening, such as by further crosslinking using a secondary PEGDA gel post-fabrication since this strategy appears to be unidirectional. If we can create stiffness-matched patient specific model LCs, it should be possible to both increase and decrease the stiffness of models.

Conclusion
We produced and pressurized models of the human LC with varying numbers of polymerizations. We recorded their displacement under increasing pressure to evaluate any change in stiffness. The results show that polymerizing LC models an additional time may increase a model’s displacement. We must test additional samples to determine whether this increase is statistically significant. Furthermore, we should evaluate other means of increasing stiffness, since this method appears to only decrease stiffness.

Acknowledgements (Optional): : This research was funded by NIH T32EY017271 Interdisciplinary Visual Sciences (IVS) Training Program, NIH R01EY020890 to Dr. Jonathan P. Vande Geest and the SURI program at the Swanson School of Engineering and the Office of the Provost at the University of Pittsburgh.

References (Optional): : [1] Quigley, H A et al., Brit J Ophthalmol, 90:262-267, 2006
[2] Crawford Downs, J et al., Exp Eye Res, 93:133-140, 2011.
[3] Strickland, R G, et al., Int J Mol Sci, 23:8068, 2022.
[4] Machiele, R, et al., StatPearls [Internet]. 2023.