Anisotropic Agarose Scaffolds as Novel Gene Activated Matrices for Mineralised Tissues

Objectives: Regeneration of gradated dental tissue interfaces requires gene delivery modalities that can provide multiple, distinct morphogenic cues in a spatially controlled manner, preferably as morphogenic gradients to moderate the transition of tissue types for interface generation. Recently, an electrophoretic platform technology has been developed to control precipitation of plasmid-DNA-loaded phosphate-salt nanoparticles within an agarose hydrogel to spatially control nucleic acid drug delivery, biomineralization and therefore transfection of progenitor cells such as dental pulp stem cells. A release study was conducted in simulated body fluid (SBF) to investigate the release of plasmid DNA (pDNA), calcium ions and phosphate ions from the loaded hydrogels.
Methods: Agarose hydrogels prepared in a phosphate-loaded buffer were sequentially loaded with pDNAs and calcium chloride solution, across a range of concentrations, using a novel electrophoretic platform to spatially control the precipitation of calcium-loaded, transfection-grade pDNA nanoparticles. Hydrogel samples were placed in SBF, incubated and sacrificed at time points. Suitable assays were used to analyse the release of pDNA, calcium ions and phosphate ions into the supernatant.
Results: Fluorometric DNA assays demonstrated that hydrogel systems with larger amounts of calcium-phosphate nanoparticles resulting from higher calcium concentration released smaller amounts of pDNA into solution. This indicates a dose-dependent complexation of the pDNA payload to calcium-phosphate nanoparticles. Meanwhile, colorimetric calcium and phosphate assays showed that an increasing amount of calcium-phosphate nanoparticles in hydrogel systems resulted in greater absorption of calcium and phosphate from solution, indicating osteoconductive and hydroxyapatite nucleating properties.
Conclusions: This release study demonstrates desirable bioactivity of biomineralized hydrogels in vitro using a relevant SBF solution. Further development of the novel electrophoretic platform will allow tuneable nucleation and growth of the pDNA-nanoparticles to optimize transfection efficiencies. Meanwhile, the progression to a 3D-electrophoretic system will allow the production of multiple-phasic hydrogel scaffolds to instruct interfacial tissue and pulp tissue regeneration.