Comparative study of decellularization techniques to obtain natural extracellular matrix scaffolds of human peripheral-nerve allografts.

BACKGROUND

We hypothesize that adding sonication cycles to the process of decellularization of cadaveric human peripheral nerves will increase the removal of cell debris and myelin sheath, increasing their utility as allografts.

METHODS

Our aim of this study was to develop a decellularization protocol that allows the removal of cells and myelin sheath without detrimental effects on nerve architecture. Segments of ulnar and median nerves from human donors, isolated both before and after cardiac arrest, were subjected to two methods of decellularization: two-detergent-based (M1) and the same method with sonication added (M2). We evaluated the histology of unprocessed and decellularized nerves (n = 24 per group) for general morphology, presence of cell nuclei, nuclear remnants, collagen fibers, and myelin. We performed immunohistochemistry to verify the removal of Schwann cells associated with histomorphometry. We used scanning electron microscopy (EM) to evaluate the ultrastructure of both native and decellularized nerves. The efficacy of decellularization was assessed by analysis of genomic DNA.

RESULTS

Histology confirmed that both decellularization protocols were adequate and maintained natural nerve architecture. Scanning EM showed that 3D ultrastructural architecture also was maintained. Histomorphometric parameters showed a more complete removal of the myelin with the M2 protocol than with M1 (p = 0.009). Fiber diameter and density were not modified by decellularization methods.

CONCLUSIONS

Sonication can be a complementary method to decellularization of peripheral nerve allografts with sonication increasing the effectiveness of detergent-based protocols for the removal of unwanted cellular components from peripheral nerve allografts.