Cell Dancing Enhances 3D Stem Cell Chondrogenesis via Early-Stage Cytoskeleton and Nuclear Mechanosensing

Introduction: Matrix compliance in 3D cellular niches, which can be introduced by molecular-level mobility, has been shown to significantly impact stem cell behavior in physically crosslinked hydrogels. However, molecular mobility is often coupled to stress relaxation in these systems, and the observed cellular behaviors are attributed to stress relaxation. To elucidate the independent role of molecular mobility on stem cell differentiation without the confounding factor of stress relaxation, our lab has previously reported PEG-based sliding hydrogels (SG) as a unique 3D stem cell niche. Unlike conventional chemical hydrogels (CG) with rigid crosslinks and ligands, SG is characterized by mobile crosslinks and ligands that can slide along the polymer chain. The mobile crosslinks in SG are based on stable covalent bonds, and therefore SG is elastic and does not exhibit stress relaxation. Compared to the non-mobile CG, the mobile SG significantly accelerates human mesenchymal stem cell (hMSC) chondrogenesis with enhanced neocartilage deposition in 3D. While our previous work highlights the importance of molecular mobility as a 3D stem cell niche parameter for enhancing stem cell chondrogenesis, the underlying molecular mechanisms remain largely unclear. The goal of this study is to elucidate the biophysical mechanisms through which SG enhances hMSC chondrogenesis by thoroughly characterizing mechanosensing pathways across multiple levels including cell-hydrogel interface, cytoskeletal, nuclear dynamics, and chromatin accessibility.

Materials and

Methods: Sliding hydrogel (SG) precursors were synthesized as we previously reported, by threading α-CD rings onto a linear PEG chain (Tong, Advanced Materials, 2016). Chemical gel (CG) precursors used were norbornene functionalized 8-arm 10 kDa PEG. RGD was incorporated for cell adhesion. To induce hydrogel crosslinking, SG and CG precursors were mixed with 1.5K PEG dithiol and LAP photo-initiator. Human MSCs were encapsulated at 10 M/ml and cultured in chondrogenic media for 3 weeks. Various small molecule inhibitors targeting cytoskeleton, chromatin condensation, and PLA2 signaling were added to the induction media for the first 3 days of differentiation. MSC chondrogenesis was characterized by day 3 PCR and day 21 cartilage matrix deposition by histology. Extracellular matrix dynamics of hMSCs were characterized using bead displacement assays. Live cell imaging (time interval- 5 minutes and duration-16 hours) was used to quantify F-Actin (lifeact-labelled MSCs) and nuclear (NucSpot Live 650) dynamics. Microrheology assays were conducted using two PEG-functionalized nanoparticles of sizes 65 nm and 500 nm. Western blots and immunofluorescence staining were performed to assess mechanosensing at day 1. Markers tested include nuclear mechanosensing (lamin A/C, H3K9me3), and chromatin condensation (H3K27me3, H3K27ac, AcK).

Results, Conclusions, and Discussions: Live cell imaging showed SG, but not CG, induced rapid “dancing” behavior and active morphological changes of hMSCs upon 3D encapsulation, with active cytoskeletal and nuclear movements. This led to a highly dynamic cell-hydrogel interface, with pericellular matrix deformations up to 2.5 µm. Microrheological measurements highlighted that at length scales of ~65-500 nm, SG was 5-10X softer compared to CG, despite the same bulk mechanical stiffness of the two groups based on unconfined compression testing, indicating that it is much easier for cells to deform pericellular matrix in SG. Immunostaining of cytoskeletal markers showed SG led to enhanced actomyosin and microtubule network formation, and 7X higher nuclear movement speeds. This was accompanied by increased nuclear mechanical loading, as indicated by Lamin A/C and H3K9me3 upregulation. The cellular “dancing” in SG was active mostly during the first 3 days after encapsulation, post which it gradually diminished. Correspondingly, inhibiting cytoskeletal and nuclear dynamics and mechanical loading for the first 24-72 hours out of a 3-week culture was sufficient to abolish the enhanced hMSC chondrogenesis in SG. Concurrently, inhibiting the cytoskeleton any time after 4 days of culture had minimal impact on cartilage formation. These results highlight that early cytoskeletal and nuclear dynamics of hMSCs in SG are critical for the observed enhanced hMSC chondrogenesis. SG also enhanced hMSC chromatin accessibility, as noted by an increase in transcription activation histone acetylation marker (AcK) and a decrease in repression marker H3K27me3 expression. Inhibiting the cytoskeleton, which blocked nuclear mechanical loading, reduced chromatin accessibility. Finally, we identified Phospholipase A2 (PLA2)- Arachidonic Acid (ARA) signaling as a transcription-independent mechanism of regulating MSC mechanosensing and chondrogenesis in SG. We observed that inhibiting PLA2 and introducing ARA for the first 72 hours of hMSC chondrogenesis moderately reduced and significantly enhanced hMSC cartilage matrix formation after 3 weeks respectively, by modulating nuclear mechanical loading. In summary, the present study identified key biophysical mechanisms through which SG enhances hMSC chondrogenesis in 3D and validated molecular mobility as an important parameter in 3D cell niche design to introduce matrix compliance and promote desirable stem cell differentiation and tissue regeneration.

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