(C-118) Controlled Nerve Regeneration Facilitated by Electrical Stimulation of Dual-Layered Microspheres

Peripheral nerve injuries can lead to loss of sensation and loss of function. Short gaps and defects can be repaired using end-to-end suturing. The gold standard treatment for large sized defects is the use of an autologous nerve graft that requires multiple surgeries and often results in incomplete recovery. Our lab focuses on biomaterial-based therapies for nerve repair. Previous studies have shown that aligned nanofibers combined with growth factors can enhance neurite growth. However, sustained growth factor delivery is still a challenge. PLGA microspheres hydrolyze in water, resulting in high initial burst release. Gelatin microspheres can maintain a controlled release but are susceptible to enzymatic degradation and the release profile is nonlinear. The goal of this project is to develop a dual-layered microsphere that allows for sustained, linear drug release that is initiated by electrical stimulation. We have fabricated a hyaluronic acid-carbon nanotube conductive nanofiber mat that is seeded with our bioengineered dual-layered PLGA-gelatin microspheres that can be controlled via electrical stimulation to maintain a linear drug and growth factor release. Electrical stimulation will activate the gelatin layer in our dual-layered microspheres to initiate the drug and growth factor release. In turn, hydrolyzation of the inner PLGA core of the microspheres will facilitate neural regeneration and ultimately allow for nerve regeneration.

Microsphere fabrication was accomplished by first making PLGA (75:25) microspheres using a water/oil/water emulsion. A gelatin layer was added to the microspheres by way of one of three methods: (1) Adsorption – dispersing PLGA microspheres into 6% gelatin type [A/B] for 24 hours, (2) Absorption – dispersing lyophilized PLGA microspheres into 6% gelatin type [A/B] for 24 hours, and (3) Chemical Conjugation – dispersing PLGA microspheres into 6% gelatin [A/B]/MES buffer, to which EDC was added to initiate conjugation between the two layers. Gelatin concentrations were analyzed via a BCA Assay and plotted against the standard absorbance to quantify the gelatin coating and concentration. The best fabrication method was determined and subsequently used for profile release testing. Drug release profiles were observed for seven days using chemical triggers (collagenase and sodium dodecyl sulfate) to imitate electrical stimulation. The selected best fit bioengineered dual-layered PLGA-gelatin microspheres were electrospun onto a hyaluronic acid-carbon nanotube (HA-CNT) nanofiber mat. Drug and growth factor release profiles were observed for fourteen days using collagenase to imitate electrical stimulation. Once determined that the fabricated microspheres seeded onto the HA-CNT nanofiber mat can effectively sustain a linear release profile, electrical stimulation will be used via a custom fabricated stimulation plate to observe drug and growth factor release. Lastly, electrical stimulation will be utilized to evaluate if our bioengineered HA-CNT nanofiber mat seeded with our dual-layered PLGA-gelatin microspheres aid in nerve ending regeneration in a cellular model.

The three methods of fabrication yielded significantly different sizes of microspheres. The average diameters of the microspheres were 78 ± 13 μm for adsorption, 89 ± 17 μm for absorption, and 54 ± 9 μm for conjugation (Figure 1). Gelatin concentration was shown to be the highest in microspheres fabricated via absorption and lowest via adsorption (Figure 2). The microspheres fabricated via conjugation yielded the strongest PLGA to gelatin interaction by way of primary amide bonds resulting in the smallest microspheres. This fabrication method was selected for subsequent drug and growth factor release testing (Figure 3). Drug release was determined by observing Methylprednisolone and BSA concentrations with the use of SDS and collagenase to act as a chemical trigger that imitates electrical stimulation. PLGA microspheres displayed an initial burst release followed by a slow drug and growth factor release. Gelatin microspheres displayed a nonlinear release profile as well. The fabricated dual-layered PLGA-gelatin microspheres displayed much more linear release profiles and mitigated burst releases in both drug and growth factor release profiles. As a result, the smart triggers used in both release profiles show that the dual-layered microspheres have a capacity for a controlled drug release by electrical stimulation.

In order to test this, the dual-layered microspheres were electrospun onto a HA-CNT nanofiber mat (Figure 4). The electrospun nanofiber mat shows that the fabricated microspheres have successfully been able to integrate into the HA-CNT scaffold which would allow for electrical stimulation to facilitate the release of both drugs and growth factors to initiate nerve regeneration. Ongoing work is focused on evaluating the bioengineered nanofiber mat seeded with dual-layered microspheres using a dorsal root ganglia nerve growth model following electrical stimulation.