An investigation into mesenchymal stromal cells’ behaviour in 3D environment of PNIPAM-based hydrogel

Mesenchymal stromal cells (MSCs) are multipotent cells, known for the ability to differentiate into cells of bone, fat and cartilage. MSCs are commonly sourced from the bone marrow environment, where these cells reside in a 3-dimensional (3D) environment and are exposed to components of the extracellular matrix (ECM), other cells types, biochemical and mechanical stimuli. Conventional monolayer culture cannot replicate the complexity of the in vivo bone marrow environment. Therefore, a more representative MSC culture environment is required.
The aim of this project was to develop a highly tunable synthetic hydrogel, on the basis of poly(N-isopropylacrylamide) (PNIPAM), to allow temperature-driven encapsulation and subsequent study of MSC behaviour in three dimensional (3D) environment. The highly branched (HB) architecture of PNIPAM polymer was obtained by means of living radical polymerisation. Further polymer functionalisation with tri-arginine peptide sequence (RRR) has stabilised hydrogel structure and reduced solvent expulsion (syneresis). Rheological studies have revealed overall resistance to deformation (G*; complex modulus) of 5wt% HB PNIPAM+RRR to be equal to 542.3 Pa at 37˚C.
MSC single cell suspensions were successfully encapsulated in HB PNIPAM+RRR 3D droplet hydrogels, demonstrating rounded morphology, absence of proliferation and stable cell viability. Differentiation potential studies of the cell-loaded hydrogels, cultured in osteogenic or adipogenic media, demonstrated osteo-conductive, osteo-inductive and adipo-inhibitive responses.
In summary, HB PNIPAM+RRR is a novel chemical entity with a thermo-responsive nature, which forms a porous and hydrated scaffold with osteo-inductive properties for MSC encapsulation at physiologically relevant temperature. HB PNIPAM is a highly functional and amenable hydrogel platform for assessment of MSC behaviour and guidance of differentiation in 3D environment.