Non-viral gene therapy for bone tissue engineering

Abstract

In bone tissue engineering bone morphogentic protein-2 (BMP-2) is one of the most commonly used growth factors. It induces stem cells to differentiate into the osteogenic lineage to form new bone. Clinically however, high dosages of protein are administered due to fast degradation, which is associated with severe side effects. By administering BMP-2 plasmid DNA instead of protein, cells can take up the gene, produce and release BMP-2 resulting in a cheap, simple and low dosed growth factor delivery method. Alginate hydrogel is used to introduce BMP-2 plasmid DNA, and acts both as a transfection agent and controlled release system, resulting in 62% transfection efficiency, and production and release from the gel 5 weeks after transfection. As a result of the transfection, osteogenic differentiation is induced in vitro and in vivo. Biphasic calcium phosphate (BCP) particles were added to the alginate gel as a core for bone formation to start on. Orthotopically no beneficial effect of BMP-2 plasmid DNA has been detected. Whereas ectopically implanted constructs combined with seeded MSCs show a positive effect of BMP-2 plasmid DNA on bone formation. Two different dosages of BMP-2 protein, a high (clinically relevant) and low (comparable to plasmid DNA-based release) dose, were implanted ectopically to compare bone formation induced by BMP-2 protein to plasmid DNA. The plasmid DNA-based constructs induced the highest amount of new formed bone and showed an earlier bone onset. This implies that BMP-2 plasmid DNA might be a future alternative for protein delivery. To take a step towards larger implants, vascularization becomes involved. To stimulate the formation of vessels and attract cells from the circulation towards the implants, gelatin micro particles containing the chemokine SDF-1α (G-SDF-1α) are added. The gelatin microparticles ensure a controlled release up to several weeks. When combined with BMP-2 plasmid DNA in alginate, no additive effect on vascularization or bone formation is achieved after 6 weeks of implantation. In the presence of seeded MSCs however the combination of BMP-2 plasmid DNA with G-SDF-1α led to an earlier bone onset and larger amount of bone formation. A different strategy to upscale our constructs is by adding 3D pores using a bioprinter. Pores are incorporated by computer controlled printing of alginate, and support the exchange of gasses and nutrients and facilitate vascularization. After optimization of the constructs, protein production, release, and osteogenic differentiation is increased in porous compared to solid printed constructs. From all previous results we conclude that we developed and optimized a BMP-2 plasmid DNA based constructs in alginate which has been able to efficiently transfect MSCs and induce osteogenic differentiation in vitro and in vivo. Bone formation was successfully induced as well, even up to amounts comparable to protein delivery. The constructs have been further optimized by combining them with SDF-1α, and introduce porosity with 3D bioprinting techniques which will help up scaling the constructs towards larger implants. However future safety and efficacy studies at orthotopic locations, will determine whether this gene delivery strategy is ready to be translated to clinical settings.