Poster 83 - High-throughput fabrication and assembly of geometrically controlled 3D-printed jammed microgels to instruct cell infiltration and response in regenerative scaffolds

Saturday, October 26, 2024

10:00 AM – 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

LC

Lily Crow

Research Intern
Oregon Health & Science University, United States

Co-Author(s)

AA

Avathamsa Athirasala

Post Doctoral Scholar
Oregon Health and Science University
Portland, Oregon, United States
Anthony Tahayeri, B.S.

Senior Research Assistant
Oregon Health & Science University/OHSU School of Dentistry/Cancer Early Detection Advanced Research (CEDAR)/Knight Cancer Precision Biofabrication Hub, United States
MA

May Anny Alves Fraga

Research Assistant
Oregon Health and Science University, United States

Primary Investigator(s)

LB

Luiz Bertassoni, D.D.S., Ph.D.

Associate Professor
Oregon Health & Science University/Cancer Early Detection Advanced Research (CEDAR)/Knight Cancer Precision Biofabrication Hub/OHSU School of Dentistry, United States

Introduction: The geometry of the microenvironment is known to influence cell migration, morphology, and lineage commitment, key factors in bone regeneration [1, 2]. In tissue engineering, advancements in hydrogel fabrication have enabled precise replication of key ECM characteristics at the nano and microscale, including microenvironment geometry [3]. These innovations have been furthered by the usage of granular micron-sized hydrogels, i.e. microgels, that are packed to assemble larger scaffolds. These scaffolds introduce much needed microporosity to enhance diffusion of nutrients and oxygen as well as support cell infiltration, spreading, proliferation, and differentiation [4]. The increased surface to volume ratios of such scaffolds enables penetrating cells to experience and respond to the individual microgel geometries and the interstitial spaces between them [3].

Established methods, like microfluidics, enable the fabrication of microgels in high-throughput, however, these techniques can be limited in their ability to precisely control geometric features beyond simple parameters, such as aspect ratios [5, 6]. Here, we establish a method of high-resolution, high-throughput, light-based bioprinting that enables us to control microgel geometry at the microscale. Ongoing studies are testing the effects of four different geometries— circle (no angles), square (four 90° angles), equilateral triangle (three 60° angles) and five-point star (five 36° angles)— on osteogenesis, vascularization, and innervation— key biological responses for the regeneration of large bone defects.

Materials and

Methods: The microgel precursor was formulated from gelatin methacryloyl (gelMA), with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator, buffered in phosphate-buffered saline (PBS). The microgels were then fabricated with a Bionova X light-based 3D-bioprinter (Cellink, BICO) according to a computer-aided design (CAD). Print settings on the 3D printer were optimized by modifying the light intensity (from 0-100%) and print speed (0.0001-0.02 mm/sec), and the microgel precursor was optimized by varying the concentrations of gelMA (5-20% w/v) and LAP (0.15-1% w/v) for all geometries, 200 µm across x 100 µm tall.

Accuracy of each microgel was assessed based on print resolution and degree of over or under curing. Percentage print accuracy was determined by overall and feature dimensions of each microgel relative to CAD measurements. Measurements were performed from wide field microscopy images using ImageJ for at least 9 prints in each geometry. Statistical significance was determined from a student’s t-test (α = 0.05) and results are shown as average ± standard deviation.

After identifying print settings and microgel formulation that consistently achieved a high degree of print accuracy across all geometries, production throughput was increased to fabricate ~500 microgels per print. These microgels were then collected and placed in a 96 well plate with a PDMS spacer 2.5 mm in diameter and 0.4 mm tall, simulating a bone defect for regeneration. The well plate was centrifuged (1500 rpm, 1 min) to achieve a high packing density. The scaffolds were visualized by 3D renderings of confocal images in constructs that contained 0.1% (w/v) methacrylated rhodamine.

Results, Conclusions, and Discussions: The optimal conditions to consistently and reproducibly fabricate microgels (200 µm x 100 µm) at high resolution were identified to be 90% light intensity with a print speed of 0.001 mm/sec using a microgel formulation of 20% (w/v) gelMA with 0.35% (w/v) LAP. Here, print accuracy and consistency was determined as < 10% deviation from CAD dimensions for both overall as well as feature dimensions for each geometry. Each microgel maintained its adherence to the print surface with minimal distortion upon stimulation, demonstrating the integrity of the prints under the optimized conditions. The approach utilized for the assembly of microgels into a single construct achieved a high packing density, evidenced by 3D imaging and comparison of pore size. The precise geometry and high packing density of the engineered scaffolds increases the influence of scaffold geometry on cell behavior allowing us to determine the optimal geometry for osteogenesis, vascularization, and innervation.

Here, we introduce a light-based bioprinting method for the fabrication of high-resolution, high-throughput microgels with microscale geometric control down to 15 µm size, with 90 ± 5% printing resolution, and as many as 600 microgels per print. This allows us to study the effect of microenvironment geometry on cell migration, morphology, and lineage commitment to identify the ideal geometry for osteogenesis, vascularization, and innervation. The findings of this work contribute to ongoing research in engineering tissue microenvironments to support tissue formation, morphogenesis and regeneration.

Acknowledgements (Optional): We would like to acknowledge the Knight Cancer Precision Biofabrication Hub and the Knight Cancer Institute’s Cancer Early Detection Advanced Research Center for making this research possible. Additionally, we would like to thank the researchers of the Biofabrication Hub for their collaboration and support. This research has been funded by NIDCR 5RO1DE026710-05 to Luiz Bertassoni.

References (Optional): 1. Min Bao, J.X., Wilhelm T. S. Huck, Recent Advances in Engineering the Stem Cell Microniche in 3D. Advanced Science, 2018. 5(8).
2. Pingping Han, G.A.G., Georg N. Duda, Sašo Ivanovski, Patrina S.P. Poh, Scaffold geometry modulation of mechanotransduction and its influence on epigenetics. Acta Biomaterialia, 2023. 163.
3. Kristopher A. Kilian, B.B., Bruce T. Lahn, and Milan Mrksich Geometric cues for directing the differentiation of mesenchymal stem cells. Proceedings of the National Academy of Sciences, 2010.
4. Andrew C. Daly, L.R., Tatiana Segura, and Jason A. Burdick, Hydrogel microparticles for biomedical applications. Nature Reviews Materials, 2019.
5. Zhenyang Wei, S.W., Jouni Hirvonen, Hélder A. Santos, Wei Li, Microfluidics Fabrication of Micrometer-Sized Hydrogels with Precisely Controlled Geometries for Biomedical Applications. Advanced Healthcare Materials, 2022. 11(16).
6. Mohamed G. A. Mohamed, P.A., Roya Samanipour, Annie Yang, Ali Ghafoor, and Keekyoung Kim, Microfluidics-based fabrication of cell-laden microgels. Biomicrofluidics, 2020.