Manufacturing nerve guidance conduits by stereolithography for use in peripheral nerve regeneration
A series of tests were conducted for this thesis to study the use of p oly(ethylene glycol) (PEG) in the construction of nerve guidance conduits via stereolithography (SL). First, it was important to test how growth factors can be integrated into a nerve guidance conduit to promote neurite extension, improving the prospect of regeneration. For this, nerve growth factor (NGF) was incorporated into PEG scaffolds through encapsulation and conjugation of the protein. The rate of release of NGF by the scaffold was tested over a 35-day period using 2 molecular weights of PEG. The bioactivity of NGF was tested by seeding PC-12 cells with it and analyzing neurite extension. Then, cells were seeded directly on a PEG surface to test if they can attach to the material through the use of a cell adhesion peptide, RGDS. In addition, NGF was added to already-attached cells to verify that neurite extension can also be accomplished on a PEG surface. These results were compared to those achieved by growing cells on tissue culture-treated plastic. In vivo tests were conducted by implanting single-lumen and multi-lumen conduits into Sprague-Dawley rats. Histomorphological analysis compared axonal regeneration in proximal, middle, and distal portions of the conduit to that found in healthy, uninjured, nerve. These tests were also used to compare the efficiency of single and multi-lumen conduits. Cell attachment to PEG may be increased if the surface area available for attachment is increased. For this, an alternative construction method was studied using a modified SL system. A commercial SL system was retrofitted in a biosafety cabinet and the optical system was changed to decrease the diameter of the laser beam. The decrease in diameter would permit construction of nerve guidance conduits of an increased number of lumens by reducing the diameter. Construction of 14, 16, and 18-lumen samples was attempted using this new, modified, SL system. The NGF proved to be bioactive in that it promoted neurite extension in PC-12 cells. When encapsulating NGF, a greater release was seen using a higher molecular weight of PEGda. On the other hand, when conjugating NGF, a greater release was seen from a lower molecular weight. A quick initial burst was present from all of the samples. RGDS did, in fact, produce attachment of cells to a PEG surface. However, the amount of cells attached was less than that seen on tissue-culture treated plastic. Addition of NGF promoted neurite extension on both surfaces with a greater percentage of cells extending neurites on the PEG surface. This is most likely due to the fact that neurites extend better on more flexible surfaces as less tension is required. In vivo tests showed that nerve regeneration can be achieved by using SL-fabricated PEG conduits. Upon histomorphological analysis, single-lumen conduits yielded results comparable to controls as seen in axon diameter, myelin thickness, and total number of fibers. On the other hand, histomorphological analysis of multi-lumen conduits could not be achieved due to problems in the staining and sectioning of the sections. Finally, when using the modified SL system, it was also possible to produce conduits with (or conduit sections) sections containing as many as 18 lumens. Calculations of samples in a swollen state showed 7-lumen conduits had a surface area of 125.6 mm2 while 18-lumen conduits had an increased surface area of 193.5 mm2 . Nerve injuries can be very debilitating and can interrupt a person's lifestyle significantly. This research studied SL as a means for constructing PEG nerve guidance conduits as an alternative to current methods of repair. As a building technique, SL offers several advantages in the design and finish of the product. Results achieved in this thesis demonstrated that PEG can be made bioactive through addition of RGDS, a cell adhesion peptide, and NGF, a protein that stimulates neurite extension. In vivo tests showed that regeneration can be achieved using single-lumen conduits. More tests are required to study regeneration using multi-lumen conduits as problems in the histological preparation hindered the analysis of such. Multi-lumen conduits (containing up to 18 lumens) can, however, be built using a laser of reduced diameter, increasing the surface area available for cell attachment. Although more tests are required, the ability to regenerate injured nerves using SL is possible. (Abstract shortened by UMI.)
Perez, Mireya Aidee, "Manufacturing nerve guidance conduits by stereolithography for use in peripheral nerve regeneration" (2013). ETD Collection for University of Texas, El Paso. AAI1551240.