Poster P7 - Degree of Hyaluronic Acid Polymer Modification Guides Nascent Protein Secretion and Cell Fate Independent of Hydrogel Mechanics

Introduction: Synthetic and semi-synthetic biomaterials and, in particular, hydrogels have shown great promise in replicating certain aspects of the extracellular matrix (ECM) in vitro, such as by incorporating specific mechanical and biochemical signals. For example, several studies have shown that adjusting engineered hydrogel mechanics and degradability is a critical parameter in guiding cellular functions, including proliferation and differentiation.
However, cells also constantly remodel their environment, including by the deposition of newly secreted (nascent) ECM. In fact, prior findings showed that this pericellular nascent ECM provides inherent biochemical and biomechanical signals, ultimately redefining the cellular microenvironment that guides cell function[1, 2]. These dynamic interactions between cells, the hydrogel and their nascent ECM challenge the traditional paradigm of direct hydrogel-induced cellular function. Still, there is a significant gap in knowledge in understanding the mechanism of how engineered hydrogel signals, cells, and nascent ECM interact with each other. To address this, our study investigated how biochemical ligand availability and wettability, affect the dynamic interplay between cells, their nascent ECM, and the hydrogel. We used engineered hyaluronic acid (HA) hydrogels with matched mechanics but varying degrees of modifications to modulate ligand availability and wettability and measure the effect on nascent ECM deposition in space and time. Using advanced metabolic labeling techniques, we tracked how nascent ECM is deposited within these modified hydrogels.

Materials and

Methods: Norbornene-modified HA polymers were synthesized with varying degrees of substitution (DS%), including Low (~10%), Medium (~20%), and High (~40%), as quantified by 1H NMR[3]. HA hydrogels were crosslinked using a light-sensitive thiol-ene reaction with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 1,4-dithiothreitol (DTT) to achieve 5 kPa elastic moduli. Juvenile bovine chondrocytes were isolated from the articular cartilage of two-month-old bovines and expanded in DMEM growth media containing 10% fetal bovine serum for one passage prior embedding into HA hydrogels.
Embedded cells were cultured in chondrogenic media supplemented with methionine analog L-azidohomoalanine (AHA), to metabolically label newly synthesized nascent ECM. Nascent ECM was visualized using fluorescent dibenzocyclooctyne-amine-488 or dibenzocyclooctyne-biotin mediated click chemistry for nascent ECM enrichment (using neutravidin beads)[4]. Isolated proteins were quantified with Bradford Protein Assay and mass spectrometry and normalized to dsDNA. EdU pulse-chase incorporation was used to measure cell proliferation, including in response to perturbation of nascent ECM-hydrogel interactions (using soluble RGD peptide, 0.5 mM). In addition, gene expression of cartilage oligomeric matrix protein (COMP) and aggrecan (ACAN) was quantified using real-time PCR with the ∆∆CT method, using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. Statistical analyses were performed using two-way ANOVA and Welch's t-test.

Results, Conclusions, and Discussions: Results
Within 7 days of culture, cells embedded in High DS% hydrogels, showed a six-fold increase in EdU incorporation (76.3%±7.5%) when compared to Low (12.6%±4.6%), and Medium DS% (21.5%±7.8%), indicating enhanced proliferation and cell division in response to High DS% (Figure 1A). Cells within all DS% hydrogels deposited nascent ECM within 7 days forming a shell around the cell body (Figure 1B). However, quantification of nascent ECM thickness showed increased ECM as a function of DS% with 2.72±1.23 µm (High DS%) to 1.21±0.54 µm (Low DS%), further suggesting a role of %DS in regulating cell function (Figure 1C). Increased nascent ECM deposition was supported by total ECM protein quantification with almost two-fold increase in High DS% (4.43 μg/μg of DNA) compared to Low DS% (2.74 μg/μg of DNA), suggesting that hydrogels with a higher DS% enhance nascent ECM formation and cell proliferation.
To test whether proliferation requires cells to interact with their nascent ECM, we next used soluble RGD to block cell-nascent ECM interactions. EdU incorporation was significantly reduced in High DS% (-30%) when compared Low DS% (-6%) hydrogels at day 7 (Figure 2A). These findings indicate that the increase in cell proliferation requires nascent ECM-interactions in high DS% hydrogels but has little effect on low %DS hydrogels. Interestingly, expression of selected chondrogenic genes (COMP and ACAN) showed significant downregulation for cells cultured in High DS% when compared to Low DS% hydrogels (Figure 2B).
These data suggest that hydrogel signals direct cellular function independent of hydrogel mechanics. Given that HA hydrogel modifications directly influence cell-HA interactions via CD44[5], our data suggest that higher DS% reduce CD44 ligand interaction which enhances cell proliferation and nascent ECM deposition but reduces chondrogenesis, perhaps due to cell de-differentiation.

Conclusion
This study established that HA biochemical signals direct nascent ECM deposition and cell fate, which depends on cell-nascent ECM interactions. Ongoing work is employing pulse-labeling and proteomics to further dissect the mechanisms of hydrogel-induced ECM production towards refining our understanding of cell-matrix interactions.

[1] Loebel+Nat. Mater., 2019.
[2] Loebel+Adv. Funct. Mater., 2020.
[3] Plaster+Carbohydr. Polym. Technol. Appl., 2023.
[4] Loebel+Nat. Protoc., 2022.
[5] Kwon+Biomaterials, 2019.

Acknowledgements (Optional):