Poster O3 - Aptamer-Functionalized In-Situ Injectable Hydrogel Formation Supported by Elastic Macroporous Matrix

Introduction: Hydrogels have been widely applied as mimetic extracellular matrix for carrying biologics that are crucial in tissue regeneration. They can be either readily synthesized before being applied to the site of regeneration, or be synthesized as an in situ injectable hydrogel during application to the site of regeneration. For hydrogels that are synthesized and loaded with biologics prior the application, the synthetic process commonly involves harsh conditions that can cause the incorporated biologics to lose their activities. Moreover, the biologics that are loaded to the synthesized hydrogel could potentially lose their bioactivities during shipping and storage. In situ injectable hydrogel can be used to solve these problems by enabling on-demand loading of biologics during formation of in situ injectable hydrogel. Despite their usefulness of maintaining the bioactivities of the biologics, in situ injectable hydrogels often face issues in control of gelation time. Rapid gelation will increase the difficulty of injection, while slow gelation will cause uncontrolled release and the loss of biologics from the site of injection. To overcome these concerns, an elastic macroporous scaffold was developed to retain and support the formation of subsequent an in situ injectable hydrogel. Moreover, the in situ injectable hydrogel was functionalized with aptamers that sequestered biologics to allow for controlled release.

Materials and

Methods: The elastic macroporous scaffold was synthesized using PEGDA through free radical polymerization coupled with cryogelation. The porous structure of the macroporous PEG scaffold was analyzed using SEM. The swelling ratio, porosity and Young’s modulus of the macroporous PEG scaffold were also investigated. Fibrin gel was chosen to be the in situ injectable hydrogel for this study. Fibrinogen was injected into thrombin-loaded macroporous PEG scaffold. The retention efficiency of fibrin gel formed inside the macroporous PEG scaffold the was determined by quantification of fibrinogen released into surrounding media. The efficacy of cell retention and proliferation inside the fibrin gel was evaluated by confocal microscopy imaging and ATP proliferation assay of smooth muscle cells (SMCs) over 24 hours, respectively. To sequester PDGF-BB, the target biologics, fibrinogens are functionalized with aptamers with affinity towards PDGF-BB. The efficiency of Fibrin-PEG matrix in controlled release of PDGF-BB were determined by quantifying the amount of PDGF-BB released into surrounding media using ELISA. The bioactivities of the PDGF-BB released from Fibrin-PEG matrix was evaluated through transwell cell migration assay. The efficacy of the PDGF-BB-loaded Fibrin-PEG matrix in enhancing SMCs proliferation inside the matrix was analyzed by confocal microscopy imaging of viability-stained SMCs over 7 days.

Results, Conclusions, and Discussions: Based on SEM images, the macroporous PEG scaffold was found to have evenly distributed and interconnected porous structures. The scaffold was capable of sequestering the injected solution. For the fibrinogen injected into the thrombin-loaded macroporous PEG scaffold, around 80% were retained and formed a fibrin gel in 1 minute. The formation of fibrin gel inside the macroporous PEG scaffold can be further confirmed by the 50% increase in mass, and decrease in porosity and swelling ratio of the scaffold. From confocal microscopy image, the Fibrin-PEG matrix showed in more SMCs attachment and retention in comparison to native porous PEG matrix. Result from ATP proliferation assay also suggested higher cell survival and proliferation in the Fibrin-PEG matrix compared to native porous PEG matrix. For Fibrin-PEG matrix without aptamer functionalization (Fn-PEG), only about 50% of PDGF-BB loaded were retained inside the matrix after allowing to release for 4 hours. For aptamer-functionalized Fibrin-PEG matrix (aFn-PEG), more than 80% of PDGF-BB were retained inside the matrix after allowing to release for 4 hours. In the profile of PDGF-BB release over time, aFn-PEG showed 40% reduction in burst release of PDGF-BB compared to Fn-PEG, and aFn-PEG continued to release additional 20% of PDGF-BB in a controlled manner over 2 weeks. The cell migration assay demonstrated that, 65% of the PDGF-BB collected from aFn-PEG after 2 weeks of release stilled retained their bioactivity. Comparing to Fn-PEG, SMCs that are loaded along with PDGF-BB in aFn-PEG are found to exhibit higher cell density and resulted in formation of dense cell network.
This work has demonstrated the feasibility of utilizing a PEG-based elastic macroporous scaffold to retain and support the formation of an in situ injectable hydrogel. The cell binding components in the injectable hydrogel enable cell attachment and proliferation. Furthermore, the aptamers incorporated into the injectable hydrogel can achieve growth factor sequestration and allow for controlled release over two weeks. This system has shown potential in cell and growth factor delivery for other applications in regenerative medicine.

Acknowledgements (Optional): Research reported in this work was supported in part by the National Institutes of Health (HL122311; AR073364).