Development of a Gene-Activated Scaffold Incorporating Multifunctional Cell-Penetrating Peptides for pSDF-1α Delivery for Enhanced Angiogenesis in Tissue Engineering Applications.

Rachael N Power,Brenton L Cavanagh,Fergal J O'Brien,James E Dixon,Caroline M Curtin

doi:10.3390/ijms23031460

Abstract

Non-viral gene delivery has become a popular approach in tissue engineering, as it permits the transient delivery of a therapeutic gene, in order to stimulate tissue repair. However, the efficacy of non-viral delivery vectors remains an issue. Our lab has created gene-activated scaffolds by incorporating various non-viral delivery vectors, including the glycosaminoglycan-binding enhanced transduction (GET) peptide into collagen-based scaffolds with proven osteogenic potential. A modification to the GET peptide (FLR) by substitution of arginine residues with histidine (FLH) has been designed to enhance plasmid DNA (pDNA) delivery. In this study, we complexed pDNA with combinations of FLR and FLH peptides, termed GET* nanoparticles. We sought to enhance our gene-activated scaffold platform by incorporating GET* nanoparticles into collagen–nanohydroxyapatite scaffolds with proven osteogenic capacity. GET* N/P 8 was shown to be the most effective formulation for delivery to MSCs in 2D. Furthermore, GET* N/P 8 nanoparticles incorporated into collagen–nanohydroxyapatite (coll–nHA) scaffolds at a 1:1 ratio of collagen:nanohydroxyapatite was shown to be the optimal gene-activated scaffold. pDNA encoding stromal-derived factor 1α (pSDF-1α), an angiogenic chemokine which plays a role in BMP mediated differentiation of MSCs, was then delivered to MSCs using our optimised gene-activated scaffold platform, with the aim of significantly increasing angiogenesis as an important precursor to bone repair. The GET* N/P 8 coll–nHA scaffolds successfully delivered pSDF-1α to MSCs, resulting in a significant, sustained increase in SDF-1α protein production and an enhanced angiogenic effect, a key precursor in the early stages of bone repair.

Highlights

Bone is a highly vascularised tissue, capable of spontaneously regenerating itself following minor injuries through a combination of neovascularisation and osteogenesis [1]
The different N/P ratios, where N/P ratio refers to the molar ratio of nitrogen residues in the vector to phosphate residues in the plasmid DNA (pDNA), were formulated by adjusting the proportion of FLH:FLR prior to complexation with pDNA
Togethe6r, of 20 these results demonstrated that glycosaminoglycan-binding enhanced transduction (GET)* N/P 8 nanoparticles were most effective for pDNA transfection, without adversely affecting cell viability

Summary

Introduction

Bone is a highly vascularised tissue, capable of spontaneously regenerating itself following minor injuries through a combination of neovascularisation and osteogenesis [1]. The current clinical gold standard in critical-sized bone defects is autograft procedures, whereby bone tissue is grafted from the patient’s iliac crest to the defect site. This results in adverse side effects, such as donor site morbidity and infection [3]. Tissue engineering strategies aim to repair critical-sized bone defects by mimicking the natural regeneration process, combining biomaterials such as scaffolds, biochemical cues, and cells to repair damaged or injured tissue [4]. Strategies combining natural polymers and calcium phosphate were found to be the most successful for bone repair in vivo [8]. The coll–nHA scaffolds will form the basis for this study, primarily used in conjunction with bone marrow-derived mesenchymal stromal cells (BM-MSCs)

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Results