Document Type

Dissertation

Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Aliasger Salem

Abstract

Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is an effective approach to overcome the shortcomings of delivery of growth factors in protein form. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold carrying polyethylenimine (PEI)-plasmid DNA (pDNA) complexes.

Two different pDNA were used: pDNA encoding platelet derived growth factor-B (PDGF-B) and pDNA encoding vascular endothelial growth factor (VEGF). The complexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and then characterized for size, surface charge, as well as in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the PEI-pPDGF-B complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system, using PEI-pPDGF-B and PEI-pVEGF complexes, was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. A different biomaterial, chitosan, loaded with copper was also evaluated in vivo.

The complexes were ∼100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. High magnification scanning electron microscopy imaging demonstrated the recruitment and attachment of BMSCs into the collagen scaffold containing PEI-pPDGF-B complexes. Confocal microscopy revealed significant proliferation of BMSCs on PEI-pPDGF-B complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the PEI-pPDGF-B complex-activated collagen scaffolds following 4 weeks of implantation when compared to the other treatment groups. Together these findings suggest that non-viral PDGF-B gene-activated collagen scaffolds effectively promote bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.

Public Abstract

Bone regeneration is critical in autogenous skeletal deficiencies, bone fractures and aging. Treatment with growth factors and recombinant human proteins is limited by a high degree of variability, limited bone formation, high cost and high physiological dosage needed. Lack of a continual supply of these proteins for a long time is also a limitation. Another alternative, viral gene therapy, is hindered due to safety concerns. One method to overcome these drawbacks is non-viral gene therapy.

Our aim was to develop an efficient non-viral gene delivery system using polyethylenimine (PEI) and plasmid DNA (pDNA) containing genes encoding for critical bone formation growth factors, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). I fabricated complexes of PEI and pDNA and loaded these particles, containing genes needed for producing bone, on a collagen platform. We inserted this bio-patch into a missing area of the bone in test animals. Cells located around the damaged area migrate into the scaffold, interact with the plasmid, the plasmid enters the cells, and the cells receive the genetic instructions to start producing proteins that enhance bone regeneration. In my experiments, the bio-patch successfully regrew bone fully enough to cover bone defects in test animals. It also stimulated new growth in human bone marrow stromal cells in culture.

Using this approach, I get local, sustained effect over a prolonged period of time without having to give continued doses of proteins. This bio-patch serves as an effective gene delivery and bone regeneration system with significant potential for clinical translation.

Keywords

publicabstract, Bone regeneration, Collagen, Genes, Growth factors, Scaffolds, Tissue engineering

Pages

xv, 152 pages

Bibliography

Includes bibliographical references (pages 132-152).

Copyright

Copyright 2015 Sheetal Reginald D'mello

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