Gene expression of human bone marrow-derived stem cells on liquid crystalline self-assembed hydroxyapatite nanorods for bone regeneration

Liquid crystal-like, low-dimensional and long-range ordered structures (LC) are found in human tissues including bones. The excellent mechanical and biological functions of natural bones entail the well-orchestrated nano-scale hydroxyapatite (HAp) crystals within collagen fibres matrix. However, the understanding of the self-assembling of HAp nanocrystals and formation of hierarchically ordered bone structure are still elusive. Our study aims to develop an in situ traceable anisotropic HAp nanorods doped with citrate and terbium (Cit/Tb-HAp NRs), investigate their self-assembling behaviour and illuminate their potential and pathway of guiding ordered osteogenesis at multi-scale in vitro. Human bone marrow-derived stem cells (hBMSCs, ScienCell, Carlsbad, US) were culture on coated coverslips. Different groups were set up based on the coating substrates, which are hydroxyapatite nanorods (HAp NRs) with or without 4 mol% terbium (Tb) doping, in short- or long-range ordered patterns (fixed by PDMS). The coverslips coated with only PDMS were utilised as the control. Three biological replicates were set for each condition. On day 21 of osteogenic differentiation in the Mesenchymal Stem Cell Osteogenic Differentiation Medium (MODM, ScienCell, Carlsbad, US), total RNA extraction was performed using a Monarch Total RNA Miniprep Kit (New England Biolabs, Ipswich, US), according to the manufacturer’s protocol. The RNA-Seq was carried out at the UCL Genomics facility. RNA concentration was measured on a Nanodrop spectrometer and 500 ng of RNA was used for cDNA synthesis and library preparation using the Kapa mRNA HyperPrep assay (Roche, Basel, Switzerland) and xGen UDI-UMI adapters (Integrated DNA Technology, Shanghai, China). Sequencing of these libraries was performed on the NextSeq 2000 (Illumina, San Diego, US) P2 flow-cell with 56bp paired-end reads.