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Tissue Engineering

Tissue Engineering - Poster Session A

Poster K16 - Biofabrication of a Load Bearing 3D Printed Human Lamina Cribrosa

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Glaucoma occurs when retinal ganglion cell (RGC) axons are damaged leading to irreversible blindless [1]. The lamina cribrosa (LC), a collagen network that mechanically supports RGC axons as they leave the eye, undergoes remodeling with glaucoma [2]. Due to remodeling, glaucomatous and non-glaucomatous eyes respond differently biomechanically to increases in intraocular pressure (IOP) that occur with glaucoma. Thus, LC response/deformation and its relationship to IOP can be used to evaluate glaucomatous damage and disease progression. LC morphology can be detected in the clinical setting with optical coherence tomography scans [3]. It would be clinically useful to make models for how a patient’s eyes would respond to increased IOP to aid in identifying at risk individuals for glaucoma or disease progression with a OCT scan. The purpose of this study was to print models of the LC from images taken ex-vivo and pressurize them to determine if they can carry a load and determine how they deform by taking image stacks. The deformation of these models could be useful in a clinical setting to predict disease progression of glaucoma.

Materials and

Methods:  A solvent cast Tecoflex sheet was placed in the custom-built bioreactor and displacement of the sheet was tracked as image stacks were taken on the custom- built 2 photon microscope as pressure increased from 5mmHg to 15mmHg, 30mmHg, and then to 45mmHg. Pressure was controlled with the ElveFlow OBI Microfluidic Flow Controller. Pressurization of the same sample was done 3 times to account for variability in the setup of the bioreactor. Then, a 80:20 PCL: gelatin electro-spun sheet with a 1.5mm diameter biopsy punched hole in it was thermoformed to the Tecoflex sheet. The displacement of the thermoformed sheet was then tracked in the same way.
The soft tissue biomechanics lab has access to ex-vivo image stacks of human LCs as shown in (figure 1). These image stacks were then segmented using a MATLAB code (figure 2) and printed in BIOINX N400 using the 2 photon microscope and a custom python script that utilizes the segmented images and controls where the laser polymerizes. The polymerization wavelength used was 780nm. BIOINX N400 was printed over the biopsy punched hole in the thermoformed sheet. The purpose of the sheet was to allow for the printed LC to be pressurized and to act as a pseudo-sclera. Immediately after printing, the LC print and sheet complex (figure 4) were placed in buffer provided in the BIOINX kit for one hour to allow the print to develop. After development, the print and sheet complex were pressurized, and the displacement was tracked (figure 3).

Results, Conclusions, and Discussions: The displacement of the Tecoflex sheet was more than the displacement of the sheet after thermoforming it and after printing on it. The displacement of the sheet with the print appears less than the displacement of previous sheets (figure 4).
Preliminary data appears to show that the sheet with the LC print displaces less than the sheet alone, which is expected. To prove that the LC print carries or experiences a load, its displacement would have to be less than the displacement of the thermoformed sheet. More data might need to be acquired to confirm this conclusion, but results suggest that this method of printing and pressurization accurately deforms printed LCs or models how an LC would deform.
Another trial with a different sheet would help determine if further conclusions can be made. Results for how the printed LC deforms compared to how the ex-vivo Human LC deformed has not been completed. This process would involve quantifying local modulus estimates based on the Law of Laplace from image stacks taken during pressurization and comparing estimates for the printed LC and human LC. This analysis would be useful to see if the mechanical properties of the printing material and the structure of the LC are similar to that of the Human LC. Additionally, if the gelatin of the BIOINX is not stiff enough for the difference between displacement of the sheet with the print and the sheet without the print to be significant, stiffening the gelatin with EDC crosslinker should be considered.
A printed model of Human LC was fabricated on a sheet, pressurized and imaged under a 2-photon microscope. Results show that thermoforming an additional sheet to the Tecoflex does increase stiffness. There is a trend toward a decrease in displacement when the sheet is printed on. It is suspected that the print does in fact hold pressure, but further work needs to be done with a second sample to obtain more representative results. We will also attempt to stiffen the print with EDC crosslinker to further decrease displacement which might make the results significant.

Acknowledgements (Optional):  This research was funded by NIH R01EY020890 to Dr. Jonathan P. Vande Geest, NIH T32EY017217 Interdisciplinary Visual Sciences Training Program, and the SURI program at the Swanson School of Engineering and the Office of the Provost at the University of Pittsburgh.

References
[1] Junhui Shen, Yuanqi Wang, Ke Yao. Protection of retinal ganglion cells in glaucoma: Current status and future, Experimental Eye Research,
Volume 205, 2021,108506,ISSN 0014-4835, https://doi.org/10.1016/j.exer.2021.108506.
[2] Abe RY, Gracitelli CP, Diniz-Filho A, Tatham AJ, Medeiros FA. Lamina Cribrosa in Glaucoma: Diagnosis and Monitoring. Curr Ophthalmol Rep. 2015 Jun 1;3(2):74-84. doi: 10.1007/s40135-015-0067-
7. PMID: 26052477; PMCID: PMC4455897.
[3] Leung CK. Diagnosing glaucoma progression with optical coherence tomography. Curr Opin
Ophthalmol. 2014 Mar;25(2):104-11. doi: 10.1097/ICU.0000000000000024. PMID: 24370973.