Controlled Shear Flow Directs Osteogenesis on UHMWPE-Based Hybrid Nanobiocomposites in a Custom-Designed PMMA Microfluidic Device.

Abstract

The combinatorial influence of a biophysical cue (substrate stiffness) and biomechanical cue (shear flow) on the osteogenesis modulation of human mesenchymal stem cells (hMSCs) is studied for bone regenerative applications. In this work, we report stem cell differentiation on an ultra high molecular weight polyethylene (UHMWPE)-based hybrid nanobiocomposite [reinforced with a multiwalled carbon nanotube (MWCNT) and/or nanohydroxyapatite (nHA)] under a physiologically relevant shear flow (1 Pa) in a custom-built microfluidic device. Using a genotypic assessment with qRT-PCR and phenotypic assessment through analysis of cytoskeletal remodelling and marker proteins, the role of shear on the progression of osteogenesis modulation has been quantitatively established with statistically significant differences between nHA-reinforced and MWCNT-reinforced UHMWPE. Early-stage (alkaline phosphatase activity at day 8), middle-stage (matrix collagenation at day 14), and late-stage (matrix calcification at day 20) events were analyzed using mRNA expression changes of a limited cell volume after microfluidic culture experiments. The conventional Petri dish culture (static) exhibited an increased osteogenesis for nanoparticle-reinforced UHMWPE, irrespective of the type of nanoparticle. The shear-mediated culture experiments resulted in noticeable differences in the degree of osteogenesis with MWCNT being more effective than nHA reinforcement. The shear-mediated osteogenesis has been attributed to the skewed cellular morphology with a higher cell adhesion (vinculin expression) on UHMWPE and nHA than that of UHMWPE and MWCNT. The signatures of the cytoskeletal changes are reflected in terms of left-to-right (L-R) chirality as well as alignment and pattern of actin fibers. Moreover, stemness (vimentin expression) was found to be decreased because of differentiation. The electrophysiological analysis using patch clamp experiments also revealed a higher inward calcium current and intracellular calcium activity for the cells grown on the UHMWPE and nHA nanobiocomposite under shear. Overall, the present study conclusively establishes the synergistic role of substrate stiffness and shear on osteogenesis of hMSCs, in vitro.