Posttraumatic nerve repair is one of the major challenges in regenerative medicine and microsurgery. Despite the recent progresses in the field of tissue engineering, functional recovery after severe nerve lesions is generally partial and unsatisfactory. Autologous nerve graft is still the best method to treat peripheral nerve lesions [1, 35], although it has several drawbacks and does not allow complete functional recovery. Extensive research has suggested that complete recovery of nerve functionality could ideally be achieved by guiding axon regeneration toward its original tissue target, using intraluminal nerve channels [6, 7, 8]. In the last decades, a wide variety of artificial nerve guides of natural or synthetic origins have been engineered to be used as bioactive fillers, porous and non-porous, biodegradables or not, combining their use with the addition of essential neurotrophic growth factors or supportive cells, some of which approved for clinical applications [10, 36, 11, 12, 7, 1, 14, 15]. The current trend is the development of biomimetic nerve guides capable of providing topological, chemotactic and haptotactic signaling to the inner cells involved in the nerve repair process. In order to enhance peripheral nerve regeneration outcomes in the presence of artificial conduits, a better understanding of the underlying mechanisms of repair is required. In this study, we investigate the use of an inverted human umbilical cord artery (iHUA) as a 3D scaffold nerve chamber for improving peripheral nerve regeneration after complete SN transection in rat.
The iHUA insert was well tolerated at the implantation site and was, on average, degraded at 80% within 8 weeks. Chamber residues did not induce any significant necrosis, and only a low degree of inflammation, combined with some edema and congestion, was observed at early stage but disappeared afterwards. Fibrosis of moderate intensity was initially detected and therefore considered as a beneficial scaffold for nerve regrowth and integration of the implant. A progressive nerve chamber integration to the tissues (cell colonization and iHUA insert degradation), concomitant to the nerve reconstruction process, was observed over 8 weeks. A progressive bridging of the transected stumps of the SN has been observed over time. On day 7, tiny newly formed axons expand in random direction from the proximal regeneration cones, embedding the outside of the chamber. After 3 weeks, axons regrowth filled the inner part of grafted area, and gradually elongate towards the distal stump, forming a dense network of regular arrangement, oriented parallel to the nerve chamber, close to the standard aspect of a nerve fascicle. After 8 weeks, the axon gap filling occupancy connecting both stumps was almost complete, demonstrating that the anastomosis is effective.
Schwann cells play a pivotal role in the selective promotion of motor and sensory axon regeneration, forming Büngner bands within nerves, hence providing a guidance substrate for the re-growth of axons of which they orchestrate the remyelination [37, 38]. They are also involved in the secretion of neurotrophic growth factors and cytokines, establishing a permissive local microenvironment for nerve regeneration [39, 40, 41, 38]. From day 7, they were seen proliferating on both extremities of the wounded SN, and on the outer part of the nerve chamber which they also started to invade. Their active expansion at the distal stump, together with the typical feature of Wallerian degeneration observed from early stage and persisting for at least 2 weeks, are compatible with their role clearing debris through phagocytosis and recruiting macrophages, and their involvement in nerve stump breakdown to give way to newly regenerating axons [42, 3]. Over time, the density of Schwann cells increased, and they gradually migrated to the center of the chamber where they got aligned to form thicker and longer glial bands covering the entire width, overlapping with that of growing axons. After 8 weeks, their distribution pattern was similar to the one observed in the intact collateral SN. However, a low yield of myelination, limited to the proximal edges or focally spread along the newly forms axons, was detected only late in 4/5 rats. These observations are in concordance with the data described in the literature, which stipulate that the myelination is generally incomplete at 8 and 12 weeks after peripheral nerve transection in rats [9]. At that time, regenerating axons supported by Büngner bands were still immature but the SN reconstruction was still in progress, with minimal signs of local fibrosis and inflammation.
Although axonal regeneration and their remyelination occur naturally in the peripheral nervous system, axons often display thinner myelin sheaths and a reduced internodal length, leading to slower nerve conduction affecting sensitivity and motricity [43]. Indeed, the first signs of leg climbing motricity recovery were observed at day 3 in 27% of the rats, with a complete recovery after 8 weeks. Moreover, reflex retraction subsequent to a pinching stimulus was detected in 47% of the animals at 5 days and the sensitivity recovery reached 80% at 2 weeks. However, weak signals of motricity monitoring the tonus of the leg in the emptiness were detected much later (day 24). Overall, the recovery of SN function was partial after 8 weeks, reaching 62% of the responses recorded for the opposite healthy hind-paw. These results matched with our assessment of the neighboring GM atrophy over time. Indeed, it is well established that denervated muscles suffer from progressive degeneration [16, 34]. We observed a rapid and severe reduction of 29% at one week and 65% at three weeks of the GM weight and 23% at week one and 65% at three weeks of the GM volume, combined with a decrease in the diameter of the muscle fibers of 42% of their original size during the first 3 weeks following the intervention. From this time, GM deterioration slow down; reduction of 77% and 74% of the GM weight and volume, respectively, and of 55% of the fibers diameter at 8 weeks; suggesting the launch of muscle innervation recovery (preparation of the 2 stumps of the injured SN, directed anastomosis with elongation of axons regrowth from the proximal to the distal end, and beginning of their progressive remyelination) confirmed by our histological observations.