Instructive Nanofibrous Scaffold Comprising Runt-Related Transcription Factor 2 Gene Delivery for Bone Tissue Engineering

Abstract

Inducer molecules capable of regulating mesenchymal stem cell differentiation into specific lineages have proven effective in basic science and in preclinical studies. Runt-related transcription factor 2 (RUNX2) is considered to be the central gene involved in the osteoblast phenotype induction, which may be advantageous for inducing bone tissue regeneration. This work envisions the development of a platform for gene delivery, combining liposomes as gene delivery devices, with electrospun nanofiber mesh (NFM) as a tissue engineering scaffold. pDNA-loaded liposomes were immobilized at the surface of functionalized polycaprolactone (PCL) NFM. Human bone-marrow-derived mesenchymal stem cells (hBMSCs) cultured on RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFM showed enhanced levels of metabolic activity and total protein synthesis. RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFMs induce a long-term gene expression of eGFP and RUNX2 by cultured hBMSCs. Furthermore, osteogenic differentiation of hBMSCs was also achieved by the overexpression of other osteogenic markers in medium free of osteogenic supplementation. These findings demonstrate that surface immobilization of RUNX2 plasmid onto elestrospun PCL NFM can produce long-term gene expression in vitro, which may be employed to enhance the osteoinductive properties of scaffolds used for bone tissue engineering strategies.

Liposomes in tissue engineering and regenerative medicine

“…To facilitate liposome adsorption, scaffold surfaces were coated with various extracellular matrix proteins, which were able to transfect a higher number of cells, while at the same time reducing the amount of DNA required [127]. Recently, a chemical modification of electrospun polycaprolactone (PCL) nanofibre meshes (NFMs) was reported enabling the immobilization of bioactive-loaded liposomes onto their surfaces [124,125]. To achieve this, initial UV-ozone irradiation was used to generate reactive free radicals that were immediately subjected to aminolysis.…”

“…These modified surfaces were reacted with 2IT to generate sulfide (SH) pendant groups. Dexamethasone and pDNA-encoding RUNX2-loaded liposomes were covalently bonded to the SH groups present at the surface of electrospun NFMs [124,125]. The availability of the drug-release vehicle at the surface of the NFMs (where initial cellular contact occurs) enables a sustainable release of the Dex in the vicinity of the cells in culture and, consequently, increases its efficacy and bioavailability [125].…”

“…The availability of the drug-release vehicle at the surface of the NFMs (where initial cellular contact occurs) enables a sustainable release of the Dex in the vicinity of the cells in culture and, consequently, increases its efficacy and bioavailability [125]. It was concluded that the amount of liposomes immobilized is specifically controlled by the amount of SH groups available at the nanofibres' surface [124,125]. Another strategy to combine liposomes and nanofibres is to use coaxial electrospinning.…”

“…2003 [143] fibronectin-coated PLG [124] rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140459…”

“…Therefore, gene therapy, encoding transcription factors or encoding for a specific or to a set of proteins may be a good approach to overcome these limitations and to control stem cell differentiation [124]. Transcription factors would ensure that expression of all natural splice variants occurs in a coordinated time and sequence, and may regulate a cascade of multiple different genes.…”