Combination of BMP2 and EZH2 Inhibition to Stimulate Osteogenesis in a 3D Bone Reconstruction Model

Abstract

High concentrations of bone morphogenetic protein 2 (BMP2) in bone regeneration cause adverse events (e.g, heterotopic bone formation and acute inflammation). This study examines novel epigenetic strategies (i.e., EZH2 inhibition) for augmenting osteogenesis, thereby aiming to reduce the required BMP2 dose in vivo for bone regeneration and minimize these adverse effects. Human bone marrow-derived mesenchymal stem cells (BMSCs) were grown on three-dimensional (3D)-printed medical-grade polycaprolactone scaffolds and incubated in osteogenic media containing 50 ng/mL BMP2 and/or 5 μM GSK126 (EZH2 inhibitor) for 6 days (n = 3 per group and timepoint). Constructs were harvested for realtime quantitative polymerase chain reaction analysis at Day 10 and immunofluorescence (IF) microscopy at Day 21. After pretreating for 6 days and maintaining in osteogenic media for 4 days, BMSC-seeded scaffolds were also implanted in an immunocompromised subcutaneous murine model (n = 39; 3/group/donor and 3 control scaffolds) for histological analysis at 8 weeks. Pretreatment of BMSCs with BMP2 and BMP2/GSK126 costimulated expression of osteoblast-related genes (e.g., IBSP, SP7, RUNX2, and DLX5), as well as protein accumulation (e.g., collagen type 1/COL1A1 and osteocalcin/BGLAP) based on IF staining. While in vivo implantation for 8 weeks did not result in bone formation, increased angiogenesis was observed in BMP2 and BMP2/GSK126 groups. This study finds that BMP2 and GSK126 costimulate osteogenic differentiation of MSCs on 3D scaffolds in vitro and may contribute to enhanced vascularization when implanted in vivo to support bone formation. Thus, epigenetic priming with EZH2 inhibitors may have translational potential in bone healing by permitting a reduction of BMP2 dosing in vivo to mitigate its side effects. While autografts are still the gold standard for bone reconstruction, tissue availability and donor morbidity are significant limitations. Previous attempts to use high concentrations of bone morphogenetic protein 2 (BMP2) have been shown to cause adverse events such as excessive bone formation and acute inflammation. Overall, the utilization of EZH2 inhibitors to modulate gene expression in favor of bone healing has been demonstrated in vitro in a tissue engineering strategy. Our study will pave the way to developing tissue engineering strategies involving GSK126 as an adjuvant to increase the effects of BMP2 for stimulating cells of interest on a three-dimensional scaffold for bone regeneration.