Associate Professor, PhD Wayne State University Detroit, Michigan, United States
Introduction: Peripheral nerve injuries usually lead to loss of sensation and ultimately loss of function. Short gap injuries and defects can often be repaired using end-to-end suturing. However, this is not the case for larger gap defects. The gold standard treatment for larger 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. Our lab has explored utilizing microspheres as a delivery mechanism to facilitate neural regeneration. 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 delivery mechanism that allows for sustained, linear drug release that is initiated and controlled by electrical stimulation utilizing microspheres. 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 allows continuous release and ultimately allows for nerve regeneration.
Materials and
Methods: Microsphere fabrication was accomplished by making PLGA (75:25) microspheres using a water/oil/water emulsion. A gelatin layer was added to the microspheres by way of three methods: (1) Adsorption – dispersing PLGA microspheres into 6% gelatin for 24 hours, (2) Absorption – dispersing lyophilized PLGA microspheres into 6% gelatin for 24 hours, and (3) Chemical Conjugation – dispersing PLGA microspheres into 6% gelatin/MES buffer, to which EDC was added to initiate conjugation between the two layers. The best fabrication method was determined and subsequently used for drug profile release testing. Drug release profiles were observed for 14 days using a smart trigger (collagenase) to imitate electrical stimulation. The bioengineered dual-layered PLGA-gelatin microspheres were electrospun onto a hyaluronic acid-carbon nanotube (HA-CNT) nanofiber mat. Drug release profiles were observed for 14 days using an enzymatic smart trigger (collagenase) to imitate electrical stimulation. Once determined that the fabricated dual-layered microspheres seeded onto the HA-CNT nanofiber mat can effectively sustain a linear release, electrical stimulation was used via a custom engineered stimulation plate to observe drug and growth factor release. Lastly, controlled electrical stimulation was used to facilitate release from our bioengineered HA-CNT nanofiber mat seeded with our dual-layered PLGA-gelatin microspheres in a cellular model. 50 mV, 75 mV, 100 mV, and 125 mV was applied across the bioengineered HA-CNT nanofiber mat via a custom stimulation plate for 5 minutes every 24 hours to evaluate nerve ending regeneration of chick dorsal root ganglia over a span of 5 days.
Results, Conclusions, and Discussions: The three methods of fabrication yielded different sizes of microspheres. The average diameters of the microspheres were 22.7 ± 1.1 μm for Adsorption, 36.2 ± 1.3 μm for Absorption, and 24.8 ± 1.3 μm for Conjugation (Figure 1). The microspheres fabricated via conjugation yielded the strongest PLGA to gelatin interaction by way of primary amide bond formation resulting in small yet stable microspheres. This fabrication method was selected for subsequent drug and growth factor release testing via enzymatic degradation. Drug release was determined by observing BSA concentrations with the use of collagenase to act as an enzymatic smart trigger that imitated electrical stimulation (Figure 2). Collagenase was added 24 hours after the microspheres were in PBS to ensure that there was full control over release after smart trigger was applied. The PLGA microspheres were subjected to hydrolysis as soon as they were added to the PBS, followed by a burst release. Gelatin microspheres displayed a nonlinear release. However, the dual-layered microspheres maintained integrity prior to application of the smart trigger and displayed a linearized release. Thus, the enzymatic smart trigger used showed that the dual-layered microspheres have the capacity for a controlled drug release via electrical stimulation. Electrical stimulation was used to control the drug release from the HA-CNT nanofiber mat. 50 mV, 75 mV, 100 mV, and 125 mV was applied across the HA-CNT nanofiber mat via a custom stimulation plate for 5 minutes every 24 hours for 14 days (Figure 3). 50 mV, 75 mV, and 100 mV applied showed linearized release profiles. At 50 and 75 mV, the release profiles were significantly lower than 100 and 125 mV. At 125 mV, a slight burst release was observed. This suggests that the release profiles we observe from our bioengineered scaffold are fully controllable for amount of release based on varying the voltage. Ongoing work is currently being done with all four voltages using a chick dorsal root ganglia nerve growth model to observe neural regeneration. Early data suggests that 100 mV is the ideal voltage for neural outgrowth, while 125 mV is too harsh on DRGs.
