Pre-neuralized biomimetic nerve conduits modified with freeze-thawed stem sells for spinal cord injury repair

English Keywords:Tissue engineering  Spinal cord injury  Biomimetic neural conduit  Stem cells  Pre-neuralization  Microenvironment

English Abstract:

  【Abstract】 Objectives: This study aims to construct a pre-neuralized biomimetic nerve conduit modified with freeze-thawed mesenchymal stem cell lysates(BNC@F-MSCs) and to investigate its therapeutic effects and underlying mechanisms in repairing complete spinal cord injury(SCI). Methods: Biomimetic nerve conduits(BNCs) with radially aligned microchannels were fabricated via directional freeze-casting using methacrylated silk fibroin(SFMA) and decellularized spinal cord extracellular matrix(d-ECM). Bone marrow mesenchymal stem cells(BMSCs) were cultured for 7d to induce pre-neural differentiation after seeded into the conduits, and subjected to three repeated freeze-thaw cycles to obtain lysate-loaded BNC@F-MSCs. In vitro experiments evaluated their effects on promoting neural differentiation of MSCs and guiding cell alignment along the oriented microchannels. A complete T10 spinal cord transection model was established in rats. After conduit implantation, animals were maintained in an SPF environment for 8 weeks. Histological assessment of the injury site was performed at week 1, while repair efficacy at week 8 was comprehensively evaluated through histological analysis of the injury site and bladder, bladder autonomic function testing, behavioral assessments, and electrophysiological measurements. Results: In vitro, the intracellular components released from BNC@F-MSCs enhanced Nestin and Tuj1 expression in BMSCs, induced elongated neuron-like processes, significantly increased average cell length, and aligned cell long axes within 0°-30° along the microchannels. In vivo, implantation of BNC@F-MSCs increased the proportion of M2-type macrophages(CD163+), reduced A1-type astrocytes(C3+/GFAP+) to (12.32±2.3)%, and increased A2-type astrocytes(S100A10+/GFAP+) to (60.62±8.50)%; The cavity area at the injury site decreased by 72.9% compared with the control group; At 8 weeks, electrophysiological testing showed nerve conduction amplitude of 166.20±18.21mV and latency of 2.72±0.60ms; Behavioral tests showed hindlimb footprint count recovered to 19.33±4.16 and bladder urine volume to 0.65±0.14mL. Conclusions: By simultaneously modulating the injury microenvironment and guiding ordered axonal regeneration, BNC@F-MSCs exhibit strong potential for promoting functional recovery after complete SCI.