GNAS1 and PHD2 short-interfering RNA support bone regeneration in vitro and in an in vivo sheep model.

Abstract

Our ability to guide cells in biomaterials for in vivo bone repair is limited and requires novel strategies. Short-interfering RNA (siRNA) allows the regulation of multiple cellular pathways. Core binding factor alpha 1 (Cbfa1) and hypoxia-inducible factor 1 (HIF-1) pathways can be modulated to direct bone formation via siRNA against guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1 (siGNAS1) and prolyl hydroxylase domain-containing protein 2 (siPHD2), respectively. We determined whether the administration of siGNAS1 and siPHD2 in mesenchymal stem cells (MSCs) promotes osteogenic phenotype, the dose-dependent effects of siGNAS1 on MSC differentiation to osteogenic phenotype, and whether the two siRNAs promote bone formation in vivo. siRNAs were administered to MSCs at Day 0, and protein expression of bone-specific markers was assessed at Days 1, 2, and 4 (n = 3/group/time point). In an in vivo model using seven sheep, chambers containing silk fibroin-chitosan (SFCS) scaffolds with siRNA were implanted over the periosteum and harvested at Days 7, 21, 36, and 70 (n = 4/group/time point, except at Day 70 [n = 2]) to assess bone formation. siGNAS1 promoted collagen I and osteopontin expression, whereas siPHD2 had no effect in vitro. Dose-dependent effects of siGNAS1 on ALP expression were maximal at Day 1 for 10 μg/mL and Day 4 for 100 μg/mL. In vivo, by Day 70, mean bone volume increased compared to Day 7 for siGNAS1-SFCS (47.8 versus 1.8 mg/mL) and siPHD2-SFCS (61.3 versus 1.5 mg/mL). Both siPHD2 and siGNAS1 support bone regeneration in vivo, whereas only siGNAS1 regulates bone phenotype in MSCs in vitro.