(A-38) Modulating Pentenoate-Functionalized Hyaluronic Acid Hydrogel Network Formation for Meniscal Fibrochondrocyte Mechanotransduction

Introduction:: 32.5 million people in the United States¹ and 654.1 million people globally² suffer from osteoarthritis (OA), a degenerative and debilitating joint disease. Meniscal injury poses significant risk for the development of knee OA.³ The meniscus is largely incapable of self-repair, necessitating the development of regenerative therapies. Resident cells of the tissue, meniscal fibrochondrocytes (MFCs), respond to biomechanical signals generated by their surrounding extracellular matrix (ECM). The ECM is a dynamic and viscoelastic network that deforms in response to cellular interactions. MFC-ECM mechanotransduction directs cell behaviors that are important for regeneration, including spreading, migration, new ECM deposition, and proliferation. Cell substrate elastic modulus controls MFC phenotype,⁴ but the effects of viscoelastic properties such as storage (E’) and loss (E’’) moduli on MFC behavior are not understood, revealing a need for an in vitro model of the meniscal ECM. The objective of this study is to develop a tunable pentenoate-functionalized hyaluronic acid (PHA)-based hydrogel system that mimics the mechanical and viscoelastic properties of the native meniscus for in vitro investigation of MFC-ECM mechanotransduction. It is hypothesized that control of the degree of pentenoate -ene group substitution (DoS) on hyaluronic acid will modulate the crosslink density and resulting mechanical and viscoelastic properties of the hydrogel, and subsequently alter MFC phenotype.

Materials and Methods:: Hyaluronic acid (HA) and pentenoic anhydride (PA) were esterified. Degree of pentenoate -ene group substitution (DoS) was modulated by increasing the molar ratio of PA to HA. DoS was determined by 1H NMR spectroscopy. Pentenoate-functionalized hyaluronic acid (PHA) was crosslinked into hydrogels (1:1 thiol:-ene molar ratio) using dithiothreitol as the crosslinker and lithium phenyl-2,4,6-trimethylbenzoylphosphinate for photoinitiation. Hydrogels were swollen in 10 mM NaCl for 24 hours, punched into discs, and then swollen in water, PBS, or DMEM+ for an additional 24 hours. For swelling ratio, mass of swollen hydrogels was divided by mass of dried hydrogels. For compressive moduli and viscoelastic response (E’ and E’’), data was collected using a TA RSA-III DMA instrument. Compressive modulus was defined as the slope of the elastic region of the stress-strain curve from 10% to 20% strain (uniaxial compression, 0.005 mm/s strain rate). For viscoelastic response, frequency sweeps were conducted (1% constant strain, frequency range of 628 rad/s to 0.01 rad/s). MFCs were seeded on the surface of hydrogels (15,000 cells/cm²) and cultured for 48 hours, then fixed (4% formaldehyde), permeabilized (0.1% Triton X-100), and stained with DAPI, Phalloidin Alexa Fluor 488 to visualize actin cytoskeleton, and paxillin to identify focal adhesions. Cells were taken from human donors aged 17-18 y/o (males, 3 donors) and 13-16 y/o (females, 3 donors). Images (15 images per DoS group per donor) were analyzed using ImageJ. Average cell area was determined by dividing the total area of Phalloidin fluorescence by the number of nuclei in each image.

Results, Conclusions, and Discussions:: Increasing the ratio of PA to HA during esterification increases the DoS of PHA. Increasing PHA DoS resulted in increased crosslink density, decreased swelling ratio in water, PBS, and DMEM+, and increased compressive modulus. The range of compressive moduli that is achieved in this study covers the range of compressive moduli published for native human menisci (50-1,000 kPa). The choice of solution (water, PBS, and DMEM+) tempered the effects of increased PHA DoS on swelling ratio and compressive modulus. This is due to osmotic deswelling that occurs in order for the system to reach an ion concentration equilibrium. Frequency-dependent response of the hydrogels, represented by storage modulus (E’) as a function of loss modulus (E’’), falls on the same profile as the corresponding values for native human meniscus. Modulating PHA hydrogel modulus (via PHA DoS) results in control of male MFC morphology and, specifically, cell spreading. This effect is tempered in female MFCs, where preliminary qualitative data indicates that female MFCs tend to spread less than male MFCs. Average surface area per cell was quantified in the male cells to be 188 ± 34 μm², 151 ± 31 μm², and 370 ± 534 μm² for 22% DoS, 46% DoS, and 90% DoS, respectively. The quantification of average surface area per cell for the female cells is currently underway. Paxillin is present in cells on substrates of low (22%), medium (46%), and high (90%) modulus. Overall, the MFC morphology results suggest that softer hydrogels promote a more chondrocyte-like morphology while more rigid hydrogels promote a more fibroblast-like morphology. In conclusion, determining the mechanism of mechanotransduction in the meniscus relies on synthesizing microenvironments that mimic the mechanical properties of the native tissue. This study provides the characterization necessary to show that this PHA hydrogel system approaches the static and dynamic mechanical properties of the native meniscus and can be used for further investigation of ECM mechanotransduction. Future directions include encapsulation of uncrosslinked HA in PHA hydrogels to increase control of viscous response and rate of stress relaxation, as well as encapsulation of MFCs in the hydrogel bulk for 3D culture.

Acknowledgements (Optional): :

References (Optional): : 1. Hochberg MC and Cisternas MG, US Bone and Joint Initiative, 2020.
2. Cui A, et al., EClinicalMedicine, 2020.
3. Primorac D, et al., Genes (Basel), 2020.
4. Andress B, et al., J Orthop Res, 2021.