Bone-stimulatory therapeutics include bone morphogenetic proteins (e.g. BMP2), parathyroid hormone, and antibody-based suppression of WNT antagonists. Inhibition of the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is both bone anabolic and osteoprotective. EZH2 inhibition stimulates key components of bone-stimulatory signaling pathways, including the BMP2 signaling cascade. Because of high costs and adverse effects associated with BMP2 use, here we investigated whether BMP2 dosing can be reduced by co-treatment with EZH2 inhibitors. Co-administration of BMP2 with the EZH2 inhibitor GSK126 enhanced differentiation of murine (MC3T3) osteoblasts, reflected by increased alkaline phosphatase activity, Alizarin Red staining, and expression of bone-related marker genes (e.g. Bglap and Phospho1). Strikingly, co-treatment with BMP2 (10 ng/ml) and GSK126 (5 μm) was synergistic and was as effective as 50 ng/ml BMP2 at inducing MC3T3 osteoblastogenesis. Similarly, the BMP2–GSK126 co-treatment stimulated osteogenic differentiation of human bone marrow–derived mesenchymal stem/stromal cells, reflected by induction of key osteogenic markers (e.g. Osterix/SP7 and IBSP). A combination of BMP2 (300 ng local) and GSK126 (5 μg local and 5 days of 50 mg/kg systemic) yielded more consistent bone healing than single treatments with either compound in a mouse calvarial critical-sized defect model according to results from μCT, histomorphometry, and surgical grading of qualitative X-rays. We conclude that EZH2 inhibition facilitates BMP2-mediated induction of osteogenic differentiation of progenitor cells and maturation of committed osteoblasts. We propose that epigenetic priming, coupled with bone anabolic agents, enhances osteogenesis and could be leveraged in therapeutic strategies to improve bone mass.

The physis is a well-established and anatomically distinct cartilaginous structure that is crucial for normal long-bone development and growth. Abnormalities in physis function are linked to growth plate disorders and other pediatric musculoskeletal diseases. Understanding the molecular pathways operative in the physis may permit development of regenerative therapies to complement surgically-based procedures that are the current standard of care for growth plate disorders. Here, we performed next generation RNA sequencing on mRNA isolated from human physis and other skeletal tissues (e.g., articular cartilage and bone; n = 7 for each tissue). We observed statistically significant enrichment of gene sets in the physis when compared to the other musculoskeletal tissues. Further analysis of these upregulated genes identified physis-specific networks of extracellular matrix proteins including collagens (COL2A1, COL6A1, COL9A1, COL14A1, COL16A1) and matrilins (MATN1, MATN2, MATN3), and signaling proteins in the WNT pathway (WNT10B, FZD1, FZD10, DKK2) or the FGF pathway (FGF10, FGFR4). Our results provide further insight into the gene expression networks that contribute to the physis' unique structural composition and regulatory signaling networks. Physis-specific expression profiles may guide ongoing initiatives in tissue engineering and cell-based therapies for treatment of growth plate disorders and growth modulation therapies. Furthermore, our findings provide new leads for therapeutic drug discovery that would permit future intervention through pharmacological rather than surgical strategies.

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