Non-viral Gene Delivery of BMP-2 Plasmid from Collagen Based Scaffolds for Bone Regeneration Therapy

  • Author / Creator
  • Significant progress in the field of bone regeneration therapy has been achieved in the last two decades. While regeneration therapies with autogenous, allografts and exogenous grafts are remaining the preferred treatments, the outcomes remained are usually sub-optimal. Gene therapy holds a great promise for bone regeneration, since the therapeutic genes can be delivered with a plasmid DNA (pDNA) that can be designed to promote bone regeneration, or to suppress mediators that inhibit bone formation. Since the delivery of free pDNA will likely be degraded in biological fluids and may not enter the cells on its own, a delivery agent or carrier will be needed in order to secure the entry of nucleic acids into the cells. Viral and non-viral carriers are the two main categories of gene delivery systems. Although, viral vectors are still the most successful one in gene therapy, the safety concerns are eliminating their clinical applications. On the other hand, non-viral vectors like cationic polymers (Polyethylenimine (PEI)) are less like to induce immune response but are not able to enhance transfection efficiency. Herein, we explored the potential and operational conditions of using low molecular weight polyethylenimines (PEIs) substituted with lipidic moieties carriers for the delivery of pDNA to bone-related cells in vitro. Among the modified PEIs synthesized in our lab, thioester-linked linoleic acid (PEI-tLA) with different levels of substitution has been the most successful candidate for pDNA delivery. We first showed that PEI1.2-tLA2 efficiently delivered pDNA to primary periosteum-derived cells (PDCs) and calvarial bone-derived cells (BDCs). After validating the delivery conditions, the delivery of BMP-2 plasmid from PEI 1.2-tLA2 to PDCs and BDCs successfully promoted the calcium deposition by the cells. We then explored the possibility of improving the transfection efficiency of PEI 1.2-tLA carriers by supplementing the complexation with a polyaspartic acid (pASP) additive. For this exploration, we used C2C12 and MC-3T3 cells which are well characterized osteogenic cell models. We found that PEI1.2-tLA10 was the most successful candidate for the delivery of pDNA to both cell lines and secondly that the pASP improved the transfection efficiency significantly for both cell models, but optimal conditions for the proposed delivery system differed between the cells.
    The loading of complexes into scaffolds by physical adsorption, electrospinning, chemical immobilization etc. may provide not only the needed signals for bone regeneration but also the physical support for tissue growth. The incorporation of the optimized complexes for both cell lines into three different collagen-based scaffolds (uncrosslinked (native), crosslinked and 3D mineralized collagen scaffolds) showed that 3D mineralized scaffolds carrying BMP-2 plasmid/PEI-tLA10-pASP complexes, induced the ALP activity in C2C12 cells, while the collagen scaffolds carrying the optimal complexes for MC-3T3 cells where the ones that enhanced the ALP activity. Finally, the investigated the incorporation and release of the optimized complexes from mono-layer and double-layer collagen and gelatin based electrospun nanofiber mats. The delivery of complexes from mono-layered fibers with high collagen concentration to C2C12 and MC-3T3 cells had a negative impact on encapsulation and transfection efficiency, while the fabrication of double-layered scaffolds that had collagen mat as a first layer and separated from the complexes was able to induce ALP activity in C2C12 cells. Overall, we concluded that complexes containing low molecular weight modified PEI with thioester-linked linoleic acid (PEI-tLA) and pASP as an additive can effectively deliver pBMP-2 to osteogenic cells and induce desired osteogenic features in 2D cultures. In addition, the design of 3D bioactive scaffolds can further improve the osteogenic activity of the cells, so these proposed therapeutic formulations could be further used for bone regeneration applications.

  • Subjects / Keywords
  • Graduation date
    Spring 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.