An important issue in peripheral nerve reconstruction and regeneration is bridging the nerve gap after nerve injury. Reconstruction using autologous nerve grafts still yields insufficient results. Various strategies are being developed to create an artificial nerve conduit which allows for regeneration superior to nerve grafting.11
One of the options is to incorporate growth factors using a microsphere delivery system. Microspheres are successfully used for delivery of various proteins. The advantage of the application of a conduit with microspheres over various cell loaded conduits is the immediate off-the-shelf availability. However, prior to clinical application, the release kinetics of a sphere containing conduit should be known. It takes 2 weeks for outgrowing axons to enter the conduit and approximately 4 weeks to traverse a 1cm gap in the rat sciatic nerve. Therefore, we focused on designing a microsphere delivery system, capable of delivering active NGF for that amount of time. In addition, we know of no study investigating the in vivo
release characteristics of NGF encapsulating PLGA microspheres. We studied the in vitro
and in vivo
release characteristics of microspheres, fabricated from four different types of polymers.
PLGA was chosen for this study, considering its previous use in various forms: nanoparticles, microspheres and layered nerve conduits.12-14
Upon contact with water, microsphere degradation profiles are initially characterized by a burst release in the first 24 hours. This initial burst is mostly due to the immediate release of the loosely attached protein on the outer surface of the microspheres and to a lesser extent diffusion.15
A longer period of steady release follows. Water uptake by microspheres initiates hydrolysis of esterbonds within the microsphere matrix, resulting in degradation with time.
It is important to take fabrication parameters into account when microsphere delivery systems are designed. They allow for more accurate tailoring of the delivery system. Our study has focused on the effect of polymer monomer ratio, as well as molecular weight on encapsulation efficiency and release profiles.
We found lower molecular weight polymers to have higher encapsulation efficiencies. This could be explained by the direct interaction of the protein with the polymer. Low molecular weight polymers have smaller polymer chains. Therefore, they have a higher number of end groups compared to high molecular weight polymers, when using the same weight. In this study we used carboxylic end groups, allowing for the positively charged NGF to have an ionic interaction directly to negatively charged carboxylic groups. This is in accordance with previous findings by Blanco, in which the encapsulation efficiency of lysozyme preferentially increases when using low, compared to high molecular weight PLGA.16
A second explanation for our observations is, while using high molecular weight polymers, the viscosity of the organic phase will increase and make it more difficult for the aqueous phase to disperse evenly. On the other hand, lower molecular weight polymers cause a reduction in the viscosity of the organic phase, thereby facilitating emulsification of the W1 phase. Entrapment of more protein is the result, since less protein is lost due to diffusion to the W2 phase.8
Our in vitro
study of four different types of spheres showed different degradation profiles, with protein release profiles between 0.6% and 1.6% of total per day for the first 35 days. Differences in release kinetics were shown to be dependent on polymer composition and molecular weight. Faster degradation rates were found when the glycolic acid composition of the polymer was increased. The hydrophylicity of glycolic acid causes more water uptake and thus faster degradation.17
In addition, we found the lowering of the inherent viscosity of the polymer to increase release rates. Again, lower viscosities are created by using lower molecular weight polymers. In their turn, lower molecular weight polymers need less time to degrade into smaller oligomers and monomers. This allows for more water uptake by the polymer matrix and thus for a faster release of NGF.18
Up to now, little is known about the actual in vivo release kinetics of NGF from PLGA microspheres. The in vivo part of our study demonstrated faster release (1.0% - 2.2% per day) curves for all types of microspheres compared to their in vitro counterparts (0.6% - 1.6% per day).
These findings indicate that extrapolation of in vitro kinetics to an in vivo situation would be inaccurate. Differences such as vascularization, the change in microenvironment, might play a role in altering the degradation profile of PLGA microspheres.
Nevertheless, subcutaneous implantation is the only approach that can mimic an in vivo
situation with the possibility to measure accurate radioactivity counts. Though, it would be preferable to measure counts in a situation where conduit and microspheres are used in a nerve grafting model, for instance the sciatic nerve model in the rat. We, however, did not choose this option because of potential variability in probe distance and autotomy in a rat model.19
Therefore, we were unable to account for adverse in vivo
effects specifically related to bridging a gap with a nerve conduit, such as leakage of microspheres from coaptation sites in our study. Previously, an in vivo
non-invasive screening method for release profiles has been validated by Kempen et al.10
This method, using scintillation probes for radioactivity counts, is similar to the one used in our study. Their findings show that the shielding of scintillation probes and maintaining a similar distance to the source is essential. The next step in the development of a growth factor releasing microsphere delivery system would be to incorporate it into a nerve conduit. We have previously investigated the effect of single and multichannel PLGA nerve conduits on peripheral nerve regeneration.9
Presently, the effect of NGF-microspheres incorporated into a nerve conduit is being evaluated in a rat sciatic nerve injury model.
In conclusion, we have found in vivo degradation profiles of microspheres to significantly differ from the in vitro release kinetics. In addition, we have shown release of bioactive NGF over a period of 23 days. Release kinetics can be tailored by altering polymer composition and molecular weight. Based on these findings, in combination with surface morphology data, we have selected microspheres fabricated from PLGA 50:50 4A to be best suited for use in a rat sciatic nerve injury model. Currently, the evaluation of the effect of NGF microspheres on peripheral nerve regeneration is ongoing.