Matrix viscoelasticity regulates 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, in turn, direct their cell lineage specification, which includes osteogenic differentiation in matrix conditions with high stiffness and viscoelasticity [2,3]. Furthermore, ECM mechanics have recently been shown to drive changes in epigenome modifier activity toward chromatin remodeling that alter gene expression and subsequent cell fate transitions [4,5]. Previous work has demonstrated that osteogenesis is regulated epigenetically through two histone demethylases, KDM4B and KDM6B, that act on H3K9me3 and H3K27me3, respectively [6]. Currently, it is unknown 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. In order to investigate this hypothesis, we employ the use of a 3D alginate cell culture platform whose physical properties (including stiffness, viscoelasticity, and adhesion ligand density) can be tuned independently. This approach facilitates the investigation of how cellular microenvironmental cues regulate changes within the epigenome during stem cell lineage specification 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 whose molecular weights varied from 35 to 250 kDa to impart differential stress relaxation properties (Fig. 1A). Additionally, low molecular weight alginate polymers were functionalized with polyethylene glycol (PEG) moieties via carbodiimide chemistry to further enhance stress relaxation, as previously described [7]. All alginate polymers used for cell encapsulation were functionalized with RGD adhesive peptide motifs to facilitate cell adhesion within the hydrogel matrices. Analysis of osteogenic differentiation was performed using qPCR to identify changes in expression between stress relaxation conditions, as well as during pharmacological inhibition of KDM4B and KDM6B with ML-324 and GSK-J4, respectively. Mechanotransduction signaling pathways were also inhibited 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. Further, gel samples were fixed, sectioned, and stained to assess changes in the expression of alkaline phosphatase (ALP), an early marker of osteogenic differentiation.

Results, Conclusions, and Discussions:

Results: hMSCs differentiate into early osteoblasts in stiff matrix conditions, with faster rates of stress relaxation enhancing differentiation (Fig. 1A). During differentiation, we have found that KDM4B and KDM6B expression increases with both substrate stiffness and viscoelasticity (Fig. 1B). Small molecule inhibition of rho-kinase, non-muscle myosin contractility, and focal adhesion kinase led to a decrease in ALP-positive cells (Fig. 1C) and gene expression of KDM4B and KDM6B (Fig. 1D). Furthermore, inhibition of KDM4B or KDM6B resulted in significant decreases in osteogenic differentiation in stiff, fast relaxing gel conditions based on staining for ALP (Fig. 1E) and gene expression data for early osteogenic markers (Fig. 1F).

Conclusion and

Discussion: These results illustrate how both matrix stiffness and viscoelasticity regulate MSC fate, and that the expression and activity of KDM4B and KDM6B is necessary for chromatin remodeling events during osteogenesis. 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 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.

References: [1] Darnell, Advanced Healthcare Materials (2017). [2] Engler, Cell (2006). [3] Chaudhuri, Nature Materials (2015). [4] Stowers, Nature Biomedical Engineering (2019). [5] Jang, Nature Biomedical Engineering (2021). [6] Ye, Cell Stem Cell (2012). [7] Nam, Biomaterials (2019).

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