Poster BB9 - Matrix Stiffness-Driven MSC Osteogenesis in 3D Hydrogels is Mediated through Nuclear Mechanosensing

Introduction: Mesenchymal stem cell (MSC) differentiation is modulated by niche cues such as matrix stiffness. However, how matrix stiffness modulates MSC osteogenesis in 3D and the underlying mechanism remains largely unclear. Previous studies have shown that increasing matrix stiffness enhances MSC osteogenesis in alginate hydrogels (Huebsch 2010), whereas all levels of stiffness inhibit osteogenesis in covalently-crosslinked hyaluronic acid hydrogels (Khetan 2013). This discrepancy suggests that the effect of matrix stiffness on MSC osteogenesis may be dependent on the mobility of the hydrogel network, as alginate is dynamically crosslinked and is viscoelastic, while HA hydrogels contain irreversible covalent crosslinks and are elastic.

Our lab has previously reported a unique PEG-based sliding hydrogel (SG) as a 3D stem cell platform with covalent crosslinks that can slide in 3D, providing mobile crosslinks without stress relaxation. Additionally, we demonstrated that SG promotes MSC osteogenesis in 3D compared to a non-mobile chemical hydrogel (CG) (Tong 2016). However, the effect of matrix stiffness on MSC osteogenesis in SG has never been investigated.

This study aims to investigate how varying matrix stiffness modulates MSC osteogenesis in 3D using different hydrogel platforms, including SG, CG, and alginate hydrogels. In addition, we aim to elucidate the mechanisms underlying stiffness-driven osteogenesis in 3D by thoroughly characterizing the mechanosensing pathways involved.

Materials and

Methods: Sliding hydrogel (SG) precursors were synthesized as we previously reported, by threading α-CD rings onto a linear PEG chain (Tong 2016). RGD was incorporated for cell adhesion. To induce hydrogel crosslinking, SG precursors were mixed with 1.5K PEG dithiol and LAP photo-initiator. To obtain SG with varying stiffness, PEG concentration was varied (4%, 6.7%, and 10%). Non-mobile chemically-crosslinked hydrogels (CG) and alginate hydrogels with a stress relaxation time of 140 seconds of comparable stiffness were included as controls. Human MSCs were encapsulated at 10 M/ml and cultured in osteogenic media for 4 weeks. MSC osteogenesis and bone formation were characterized using alkaline phosphatase (ALP) staining at week 1, alizarin red s (ARS) staining at weeks 2 and 4 for SG and CG, and Von Kossa staining at weeks 2 and 4 for alginate. To assess the ability of MSCs to reorganize the hydrogel network in 3D, a bead displacement assay was performed at day 0. Western blots and immunofluorescence staining were performed to assess mechanosensing at day 1. Markers tested include focal adhesions (paxillin), cytoskeletal state (f-actin, myosin IIb), nuclear mechanosensing (lamin A/C, H3K9me3), and osteogenesis transcription factors (RUNX2). Live cell imaging was conducted to assess cell and nuclear dynamics at day 0, and a custom Python script was employed to assess nuclear loading marker localization.

Results, Conclusions, and Discussions: Unconfined compressive testing showed SG with varying PEG concentrations yielded hydrogels with tunable stiffness (4.5 kPa, 11 kPa, and 21 kPa). CG and alginate controls with comparable stiffnesses were obtained. ALP staining revealed mobile SG significantly enhanced osteogenesis compared to nonmobile CG. Unexpectedly, reducing stiffness in SG promoted osteogenesis and mineralized bone formation. Consistent with previous reports, non-mobile CG showed minimal osteogenesis regardless of stiffness, whereas increasing stiffness increased osteogenesis in alginate hydrogels (Fig. 1).

Bead displacement imaging showed SG significantly enhanced matrix reorganization by MSCs in 3D compared to CG, and this effect is even more pronounced at lower stiffness levels (Fig. 2A). Soft SG (4.5 kPa) exhibited the highest MSC osteogenesis and greatest matrix reorganization. Western blot showed a similar trend (Fig. 2B). Reducing SG stiffness led to higher levels of ALP and RUNX2, along with an increased level of mechanosensing markers across all levels. Immunofluorescence staining for F-actin (red) and myosin IIb (green) further verified enhanced cytoskeletal mechanosensing in SG with decreased hydrogel stiffness (Fig. 2C). Live cell imaging of nuclear dynamics revealed that cells in soft SG displayed a significantly higher mean nuclear speed, increased lamin A/C and H3K9me3 intensity, and marker localization to the nuclear periphery (Fig. 2D). These results indicate enhanced MSC osteogenesis in SG is associated with enhanced nuclear mechanical loading and chromatin condensation. While an opposite trend was observed in how stiffness drives osteogenesis in alginate hydrogels, we observed the same correlation of enhanced lamin A/C and H3K9me3 expression along with enhanced osteogenesis (Fig. 3).

In summary, we identify nuclear mechanosensing as the conserved key driver for modulating stiffness-mediated osteogenesis in 3D. These findings help resolve the seemingly conflicting trends reported in the literature, highlighting that nuclear mechanical loading, rather than bulk matrix stiffness, predicts MSC osteogenesis in 3D. Our results also provide strong evidence to motivate future studies on using small molecules that target nuclear mechanosensing as a novel therapy to enhance MSC osteogenesis and bone regeneration in vivo.

Acknowledgements (Optional): The authors would like to thank the following funding sources: Bio-X USRP (G.M.), SGF Fellowship (M.A.), NIH R01DE024772 (F.Y.), NIH R01AR074502 (F.Y.).