IADR Abstract Archives
Non-viral Gene Delivery Using Biomimetically-mineralized Matrices for Bone Tissue Engineering
Objectives: Development of synthetic materials for bone tissue engineering will alleviate the issue of rapidly increasing demand for bone grafts. Our hypothesis is that combining calcium phosphate coatings and non-viral gene delivery in a single scaffold will further enhance bone regeneration.
Methods: 3D collagen scaffolds were cut from Collagen Plugs, and the mineralization process was carried out for 30 days. During the mineralization process, Polyethyleneimine (PEI)-DNA complexes were incubated with SBF to co-precipitate complexes into the mineral layer (CaP-GAM). Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used to characterize the calcium phosphate coating. PEI-DNA complexes were fluorescently labeled, and their location in the calcium phosphate layer was determined using confocal microscopy. Bone marrow stromal cell viability was determined using an MTS assay at 24 hrs post-seeding. Osteogenic markers were quantified using ELISA for protein production and qPCR for osteocalcin and alkaline phosphatase on days two and seven of cell culture, respectively. Five-millimeter diameter defects were created in the cranial bone of Fisher 334 rats. After implantation, scaffolds were imaged using micro-CT and visualized through H&E staining.
Results: SEM showed plate-like calcium phosphate crystal growths on the collagen, and EDS analysis revealed a calcium phosphate ratio of 1.5 which is similar to bone mineral. The apparent formation of two distinct growth factor layers suggests a biphasic release of growth factors may be achieved based upon the confocal images. Rat calvarial defects treated with the CaP-GAM contained significantly more bone compared to collagen scaffolds based on micro-CT analysis. Additionally, histology revealed new bone formation was present throughout the core of the mineralized GAM.
Conclusions: This work suggests mineralization and incorporation of gene therapeutic agents in a collagen scaffold enhanced new bone formation over the collagen scaffold alone. This work provides the basis for future investigations of CaP-GAM in bone tissue engineering.
Methods: 3D collagen scaffolds were cut from Collagen Plugs, and the mineralization process was carried out for 30 days. During the mineralization process, Polyethyleneimine (PEI)-DNA complexes were incubated with SBF to co-precipitate complexes into the mineral layer (CaP-GAM). Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used to characterize the calcium phosphate coating. PEI-DNA complexes were fluorescently labeled, and their location in the calcium phosphate layer was determined using confocal microscopy. Bone marrow stromal cell viability was determined using an MTS assay at 24 hrs post-seeding. Osteogenic markers were quantified using ELISA for protein production and qPCR for osteocalcin and alkaline phosphatase on days two and seven of cell culture, respectively. Five-millimeter diameter defects were created in the cranial bone of Fisher 334 rats. After implantation, scaffolds were imaged using micro-CT and visualized through H&E staining.
Results: SEM showed plate-like calcium phosphate crystal growths on the collagen, and EDS analysis revealed a calcium phosphate ratio of 1.5 which is similar to bone mineral. The apparent formation of two distinct growth factor layers suggests a biphasic release of growth factors may be achieved based upon the confocal images. Rat calvarial defects treated with the CaP-GAM contained significantly more bone compared to collagen scaffolds based on micro-CT analysis. Additionally, histology revealed new bone formation was present throughout the core of the mineralized GAM.
Conclusions: This work suggests mineralization and incorporation of gene therapeutic agents in a collagen scaffold enhanced new bone formation over the collagen scaffold alone. This work provides the basis for future investigations of CaP-GAM in bone tissue engineering.
