Amniotic fluid stem cell-conditioned media as a therapeutic adjunct in peripheral nerve injury repair: insights from a sciatic nerve mouse model.

Background: Peripheral nerve injuries can result in severe and lasting morbidity, including motor weakness, pain, and functional deficits, even with timely intervention. Treatment options include non-operative management and surgical interventions, such as direct end-to-end nerve repair for low-tension injuries or grafting techniques (e.g., autograft, allograft, and tissue-engineered grafts) for large-gap injuries. Recent research has focused on the regenerative potential of human amniotic fluid stem cells (AFS) due to their paracrine effects. Amniotic fluid stem cell-conditioned media (AFS-CM) has emerged as a promising therapeutic adjunct, offering ease of formulation and low immunogenicity. However, the role of AFS-CM in peripheral nerve repair remains poorly understood. This study investigates the effects of AFS-CM on peripheral nerve recovery in a sciatic nerve injury model in CD1 mice. Methods: Thirty-six male CD1 mice underwent sciatic nerve transection of the left hindlimb and were divided into three groups: (I) control group-direct end-to-end nerve repair; (II) hydrogel group-reconstruction with a hydrogel-coated silicone graft; and (III) AFS-CM group-reconstruction with an AFS-CM-infused hydrogel-coated silicone graft. Post-surgical treatments were administered biweekly via hydrogel (control and hydrogel groups) or hydrogel plus AFS-CM (AFS-CM group). Functional recovery was assessed through gait analysis, electromyography (EMG), and nerve conduction studies at two weeks, one month, and two months. Muscle and nerve tissues were analyzed using immunohistochemistry. Statistical analyses included analysis of variance (ANOVA) and generalized linear models, with a significance threshold of P<0.05. Results: No significant differences were observed in EMG amplitude and latency or the G-ratio across all groups (P>0.05). Gait analysis revealed substantial improvements in overlap distance (P<0.001) and ataxia coefficient (P=0.01) in the AFS-CM group compared to the hydrogel group. The AFS-CM group also demonstrated reduced expression of muscle RING-finger protein-1 (MURF-1), indicative of less muscle atrophy, and increased expression of alpha-bungarotoxin, suggesting improved neuromuscular junction (NMJ) recovery. Among the groups, no significant differences were noted in malondialdehyde expression, a marker of oxidative stress. Conclusions: AFS-CM shows promise as a therapeutic adjunct in peripheral nerve repair, improving functional outcomes and reducing muscle atrophy compared to hydrogel-only reconstruction. While electrophysiological and morphological outcomes showed no significant differences, the enhanced gait performance and NMJ recovery observed in the AFS-CM group highlight its regenerative potential. Further studies are warranted to elucidate the underlying mechanisms and to optimize the clinical application of AFS-CM in peripheral nerve injury management.