Date of Award
Doctor of Philosophy (PhD)
Many biological processes depend on concentration gradients in signaling molecules. Thus, introduction of spatial patterning of proteins, while retaining activity and releasability, will be critical for the field of regenerative medicine. In particular, the area of nerve regeneration is in need of innovations to improve outcomes. Only about 25% of surgical patients with peripheral nerve damage (~200,000 surgical interventions performed each year) regain full motor function with less than 3% regaining sensation. The use of nerve guidance conduits (NGC’s) which are filled with biomimetic scaffolds is one treatment being explored. These scaffolds, however, lack the spatial patterning of proteins found in native tissue. Glial-cell line derived neurotrophic factor (GDNF), a potent stimulator of axon regeneration, is one such protein that, if contained within the scaffold and conformed to a particular concentration profile, could greatly enhance neural regeneration. The object of this work is to utilize poly(ethylene glycol) (PEG) microspheres to accomplish this spatial patterning of GDNF and apply it to NGC’s.
First, an approach utilizing the controllability of the PEG microsphere’s density (buoyancy) was explored. By creating the microspheres under varying conditions, incubation time and temperature, the cross-linking and, thus, the swelling rate of the microspheres could be controlled. This created microspheres of different densities that, upon centrifugation, would orient themselves within a scaffold, creating a gradient in the different microsphere types. GDNF loaded into a batch of microspheres would thusly be oriented within the scaffold along with that particular microsphere batch. Through this, gradients in GDNF were created. Heparin was also added to the microspheres to allow for reversible binding of GDNF.
Next, gradients in reversibly bound GDNF were formed through sequential centrifugation of microsphere batches. For instance, a layer of GDNF loaded microspheres were formed into a scaffold followed by a layer of microspheres without GDNF on top of them. This created an initial step gradient in GDNF that, given time to release, would form a linear concentration gradient. Gradients formed by this method were visualized by fluorescent confocal microscopy and compared to Fickian models. Some conditions yielded profiles more linear than the model predictions, which persisted for over a week.
Lastly, the sequential gradient formation was modified and applied to NGC’s. Before the scaffolds were ready for in vivo implantation, functionalities such as cell initiated degradability, cell adhesion, and inter-microsphere cross-linking were added. A plasmin degradable peptide sequence (GCGGVRNGGK) was incorporated into the microspheres. CLICK agents, laminin, and heparin (via a new binding chemistry) were attached to the microspheres to add inter-microsphere cross-linking, add cell adhesion, and heparin binding functionalities, respectively. GDNF gradient formation and activity retention were confirmed with these fully functionalized microspheres. Microsphere scaffolds with linear gradients in GDNF were then formed in silicone tubes which were transplanted into rats with severed sciatic nerves.
Donald L Elbert
Shelly Sakiyama-Elbert, Matthew Wood, Dennis Barbour, Jin-Yu Shao