Speaker
Description
Introduction: Biological tissues exhibit intricate spatial variations, stiffness gradients, and complex niche environments critical to their biological function and implicated in various pathologies. Organ-on-chip technologies offer advanced biomimetic culture conditions compared to traditional culture methods[1-2]. However, replicating native tissue complexity remains challenging, requiring techniques to spatial patterning biomaterials within chips. This study introduces translatable micromanufacturing and biofabrication techniques to create controllable, tissue-like constructs for organ-on-chip platforms using interpenetrating polymer networks (IPNs). Techniques include digital light processing (DLP) bioprinting, extrusion-based bioprinting, and grayscale photomask gradients. Muscle-tendon and bone-cartilage interface models are used as exemplars of this spatial patterning
Methods: A gelatin-based IPN biomaterial library was developed, integrating methacryloyl and thiol-ene chemistries to modulate mechanical properties, macromer densities, and binding sites for tethered growth factors and ligands. Gelatin biomaterials were functionalised using established methods to achieve gelatin methacryloyl (GelMA; 40 and 80% degrees of functionalisation - DoF)[3-4], gelatin norbornene (GelNB; 27% DoF)[5-6], and thiolated gelatin (GelSH, 0.368 mmol/g thiol content). Precise control over synthesis parameters, including macromer concentration, molar ratios of thiol and alkene groups, norbornene/thiolated gelatin or multi-arm polyethylene glycol-norbornene (PEG-NB) or thiol (PEG-SH), and crosslinkers (dithiothreitol – DTT, photoinitiators – LAP and Igracure), produced IPNs with defined stiffness gradients in response to UV light (λ365 and 405 nm). Comprehensive characterisation involved rheological analysis, bioprinting parameters, swelling studies, mechanical assessment for localised stiffness, and bulk compression testing in confined and free-swelling conditions.
Results: Preliminary findings demonstrated GelMA’s inability to crosslink within PDMS chips due to oxygen inhibition, resulting in incomplete photopolymerisation. To address this, thiol-ene chemistry was employed using GelSH or GelMA with DTT or multi-arm PEG crosslinkers. Incorporating PEG-4SH into GelMA enabled rapid crosslinking (<1 minute) and increased the storage modulus (2.7kPa) compared to 5% (w/v) GelMA control (1.4kPa). Similarly, 5% GelSH combined with PEG-8NB exhibited rapid crosslinking (~30 seconds) and an increased modulus (14kPa), illustrating tuneability of IPNs. Initial extrusion-based studies demonstrated multi-printhead extrusion with GelMA concentrations of 7.5%, 10%, and 15% to produce constructs featuring ~two-fold stiffness gradients. Coaxial printing provided an alternative approach to fabricate gradients by modulating GelMA concentration or DoF through precise control of extrusion speed and pressure.
Discussion: Our findings demonstrate the versatility of the gelatin-based IPN library in controlling physiochemical properties. Ongoing studies are characterising IPN combinations and evaluating the influence of multi-arm PEG-NB or PEG-SH on cell behaviour, mechanical properties, and bioprinting assessment. Preliminary pilot studies using 10% (w/v) PEGDA with grayscale DLP photomasks have shown effective stiffness modulation through gradient tuning, with gelatin-based materials planned for further assessment. These approaches will be optimised for integration across diverse organ-on-chip platforms, including closed-channel systems, open-chamber designs, and custom-fabricated chips. By tailoring micromanufacturing techniques to overcome chip-specific limitations like oxygen inhibition, this work enables reproducible fabrication of tissue gradients and interfaces using a thiol-ene chemistry, improving the predictive capabilities of organ-on-chip models.
References:
[1] Ingber,D.E.Nat Rev Genet,(2022).10.1038/s41576-022-00466-9
[2] Lin,C.-C,et al.Macromol Biosci,(2024),10.1002/mabi.202300371
[3] Loessner,D,et al.Nat Protoc,(2016),10.1038/nprot.2016.037
[4] Zhu,M,et al.Sci Rep,(2019),10.1038/s41598-019-42186-x
[5] Munoz,E,et al.Biomater Sci,(2014),10.1039/c4bm00070f
[6] Soliman,B,et al.Adv Healthcare Mater,(2022),10.1002/adhm.202101873
[7] Hipwood,L,et al.Gels,(2022),10.3390/gels8120821