Matrix stiffness and viscoelasticity regulate osteogenesis through histone demethylases KDM4B and KDM6B.

Introduction: : Matrix stiffness and viscoelasticity are both known to regulate mesenchymal stem cell (MSC) differentiation.1 MSCs are able to sense these biophysical cues through mechanotransduction signaling pathways that direct cell lineage specification, which includes osteogenic differentiation in matrix conditions with high stiffness and viscoelasticity.2,3 Osteogenesis has long been understood to be regulated through canonical bone morphogen protein (BMP) signaling via BMP receptor phosphorylation of SMAD 1/5/8 transcription factors, which translocate to the nucleus and facilitate early osteogenic gene expression.4 Intriguingly, emerging evidence has demonstrated that bone morphogen protein (BMP) signaling is endogenously upregulated in MSCs cultured in stiff hydrogel conditions.5,6 Furthermore, ECM mechanics have recently been shown to drive changes in epigenome modifier activity toward chromatin remodeling events that alter gene expression and subsequent cell fate transitions.7,8 Previous work has illustrated that osteogenesis is regulated epigenetically through two histone demethylases, KDM4B and KDM6B, that act on H3K9me3 and H3K27me3, respectively, with the expression of these demethylases being regulated by the BMP-SMAD 1/5/8 signaling axis.9 However, it remains uncertain whether KDM4B or KDM6B activity is regulated by mechanotransduction signaling during osteogenesis. We hypothesize that KDM4B and KDM6B are modulated through mechanosignaling to drive osteogenic differentiation in response to matrix stiffness and viscoelasticity. To investigate this hypothesis, we employ the use of a 3D alginate hydrogel cell culture platform whose physical properties (including stiffness, viscoelasticity, and adhesion ligand density) can be tuned independently. This approach facilitates investigation of how cellular microenvironmental cues regulate changes within the epigenome during stem cell differentiation events.

Materials and

Methods: : Primary human MSCs (hMSCs) were encapsulated in alginate hydrogels and cultured for 7 days. Calcium concentration was varied to form stiff (E = 20 kPa) and soft (E = 3 kPa) matrices using alginate polymers with stress relaxation times of 20 s (fast), 200 s (medium), and 1000 s (slow). All alginate polymers used for cell encapsulation were functionalized with RGD adhesive peptide motifs to facilitate cell adhesion within the hydrogel matrices. Additionally, low molecular weight alginate polymers were functionalized with polyethylene glycol (PEG) moieties via carbodiimide chemistry to further enhance stress relaxation, as previously described.10 Gel samples were fixed, sectioned, and stained to assess changes in the expression of alkaline phosphatase (ALP), an early marker of osteogenic differentiation. Further, analysis of osteogenic differentiation was performed using qPCR to identify changes in gene expression between stress relaxation conditions, as well as during pharmacological inhibition of KDM4B and KDM6B using ML-324 and GSK-J4, respectively. Mechanotransduction signaling pathways were also perturbed using Y-276732, blebbistatin, and PF-57328 to inhibit rho-kinase (ROCK), non-muscle myosin II (NM2) contractility, and focal adhesion kinase (FAK), respectively, to assess the impact on osteogenesis. Additionally, BMP Receptor Type 1 (BMPR1) phosphorylation of SMAD 1/5/8 was pharmacologically inhibited using LDN-193189. Gel sections were stained using antibodies targeting phospho-SMAD 1/5/8 (pSMAD 1/5/8), Lamin A/C, KDM4B and KDM6B for immunofluorescence imaging.

Results, Conclusions, and Discussions:: hMSCs differentiate into early osteoblasts in stiff matrix conditions, with faster rates of stress relaxation enhancing differentiation based on staining for ALP (Fig. 1A). During differentiation, we found elevated gene expression of canonical osteogenic markers ALP, Collagen 1a1, and Osteocalcin, in addition to increased expression of both KDM4B and KDM6B in stiff, fast-relaxing matrices (Fig. 1B). Small molecule inhibition of ROCK, NM2, and FAK led to a decrease in ALP-positive cells (Fig. 2A), as well as decreases in gene expression of KDM4B and KDM6B (Fig. 2B). Additionally, inhibition of KDM4B or KDM6B resulted in significant decreases in osteogenic differentiation in stiff, fast relaxing gel conditions based on staining for ALP (Fig. 2C). Immunostaining for pSMAD 1/5/8 revealed the highest staining intensity in stiff, fast-relaxing matrices (Fig. 3A). Furthermore, subsequent inhibition of BMPR1 activity led to a marked decrease in gene expression of early osteogenic markers RUNX2 and ALP, as well as KDM4B and KDM6B (Fig. 3B).

These results illustrate how both matrix stiffness and viscoelasticity regulate MSC fate, and that SMAD pathway activity is upregulated in stiff, viscoelastic matrices. Furthermore, our results indicate that the expression and activity of KDM4B and KDM6B is necessary for chromatin remodeling events during osteogenesis. Thus, this research aims to better understand how ECM mechanics are linked to epigenetic regulation and how knowledge of these pathways can be leveraged to guide stem cell fate decisions in a coordinated manner toward applications in both disease modeling and regenerative medicine. To that end, this work strives to elucidate the biophysical mechanisms underlying bone regeneration, with the aim of identifying optimal parameters for designing biocompatible bone engraftments for fracture repair in both large wounds and patients with decreased osteogenic potential.

Acknowledgements and/or References (Optional)::

References: [1] Darnell, Advanced Healthcare Materials (2017). [2] Engler, Cell (2006). [3] Chaudhuri, Nature Materials (2015). [4] Rahman, Bone Research (2015). [5] Li, Biomacromolecules (2024). [6] Görlitz, Communications Biology (2024). [7] Stowers, Nature Biomedical Engineering (2019). [8] Jang, Nature Biomedical Engineering (2021). [9] Ye, Cell Stem Cell (2012). [10] Nam, Biomaterials (2019).