Sustained Localized Presentation of RNA Interfering Molecules From in Situ Forming Hydrogels to Guide Stem Cell Osteogenic Differentiation

Abstract

To date, RNA interfering molecules have been used to differentiate stem cells on two-dimensional (2D) substrates that do not mimic three-dimensional (3D) microenvironments in the body. Here, in situ forming poly(ethylene glycol) (PEG) hydrogels were engineered for controlled, localized and sustained delivery of RNA interfering molecules to differentiate stem cells encapsulated within the 3D polymer network. RNA interfering molecules were released from the hydrogels in a sustained and controlled manner over the course of 3-6 weeks, and exhibited high bioactivity. Importantly, it was demonstrated that the delivery of siRNA and/or miRNA from the hydrogel constructs enhanced the osteogenic differentiation of encapsulated stem cells. Prolonged delivery of siRNA and/or miRNA from this polymeric scaffold permitted extended regulation of cell behavior, unlike traditional siRNA experiments performed in vitro. This approach presents a powerful new methodology for controlling cell fate, and is promising for multiple applications in tissue engineering and regenerative medicine.

Keywords: Biomaterials; Bone regeneration; Gene delivery; Mesenchymal stem cells; Tissue engineering.

Figure 1

Synthesis of (a) 8-arm-PEG-MAES and (b) 8-arm-PEG-A.

Figure 2

¹H NMR spectra of (a) 8-arm-PEG-MAES and (b) 8-arm-PEG-A.

Figure 3

a) Swelling, b) degradation profiles and c) rheological properties of the hydrogels. d) Release profiles of siRNA/PEI complexes from the hydrogels. e) Schematic figure of siRNA/PEI complex incorporation into the hydrogels. f) Bioactivity of siRNA/PEI released from M, MA and A gels compared to that of fresh siRNA/PEI complexes and no siRNA control. *p<0.05 compared with the no siRNA control group.

Figure 4

a) Schematic figure depicting RNA and hMSCs encapsulation into the hydrogels and subsequent hMSC differentiation in osteogenic media (OM). b) Noggin gene expression, c) ALP activity, d) Runx2 gene expression, e) BSP gene expression and f) PPAR-γ gene expression of hMSCs encapsulated within the hydrogels. *p<0.05 compared with control, **p<0.05 compared with siNoggin and cotransfection, ***p<0.05 compared with cotransfection, and #p<0.05 compared with miRNA-20a at specific time point.

Figure 5

a) Calcium content in hydrogels. *p<0.05 compared with control, **p<0.05 compared with siNoggin and cotransfection, and ***p<0.05 compared with cotransfection at specific time point. All groups exhibited a significant (p<0.05) increase in calcium content over time. Mineralization in b) bulk and c) sectioned (day 14) hydrogels stained with Alizarin red. The scale bars indicate 200 μm.

Figure 6

a) ALP activity and b) calcium content in hMSC (2^nd donor)-hydrogel constructs. *p<0.05 compared with control, **p<0.05 compared with siNoggin, and ***p<0.05 compared with siNoggin and cotransfection at a specific time point.