Rat Sciatic Nerve Defect Research Articles

Muscle Grafts with Doxorubicin Pretreatment Produce "Empty Tubes" in the Basal Laminae, Promote Contentious Maturation of the Regenerated Axons, and Bridge 20-mm Sciatic Nerve Defects in Rats.

We newly developed a muscle graft that employs a doxorubicin pretreatment technique. The aims of this study were to reveal the biological and morphological features of the muscle tissue in the second week (Study I), to reveal the regeneration outcomes of functional and kinematic assessments of longer-term follow-up (16 weeks, Study II), and to make assessments of the muscle graft with doxorubicin pretreatment in the critical-sized nerve defect model (20 mm, Study III). A total of 26 adult rats were used in this study. Doxorubicin treatment was accomplished by immersion in a doxorubicin solution for 10 minutes followed by a rinsing procedure. The rats were divided into three groups: the muscle graft with and without doxorubicin pretreatment (M-graft-w-Dox and M-graft-w/o-Dox) groups and the autologous nerve graft (N-graft) group. Assays of apoptosis, immunofluorescent histochemistry including CD68 (macrophage marker), scanning electron microscopy (SEM), morphometrical studies of the regenerated axons, nerve conduction studies, and kinematic studies were performed. The M-graft-w-Dox group contained significantly larger numbers of apoptotic cells and CD68-positive cells. SEM revealed the existence of the basal lamina, so called "empty tubes," in the M-graft-w-Dox group. Study II showed contentious maturation of the regenerated axons, especially in the compound muscle action potentials. Study III showed that even at 20 mm, the M-graft-w-Dox group promoted axonal regeneration and functional regeneration. The M-graft-w-Dox group showed superior regeneration results, and this easy and short-term procedure can expand the muscle graft clinical indication for the treatment of peripheral nerve defects.

67. Silk-silk Conduits Filled with Native Spider Silk Fibers Successfully Promoted Nerve Regeneration in a 10 Mm Sciatic Nerve Defect in Rats

67. Silk-silk Conduits Filled with Native Spider Silk Fibers Successfully Promoted Nerve Regeneration in a 10 Mm Sciatic Nerve Defect in Rats

Electrodeposition of chitosan/graphene oxide conduit to enhance peripheral nerve regeneration

Currently available commercial nerve guidance conduits have been applied in the repair of peripheral nerve defects. However, a conduit exhibiting good biocompatibility remains to be developed. In this work, a series of chitosan/graphene oxide (GO) films with concentrations of GO varying from 0-1 wt% (collectively referred to as CHGF-n) were prepared by an electrodeposition technique. The effects of CHGF-n on proliferation and adhesion abilities of Schwann cells were evaluated. The results showed that Schwann cells exhibited elongated spindle shapes and upregulated expression of nerve regeneration-related factors such as Krox20 (a key myelination factor), Zeb2 (essential for Schwann cell differentiation, myelination, and nerve repair), and transforming growth factor β (a cytokine with regenerative functions). In addition, a nerve guidance conduit with a GO content of 0.25% (CHGFC-0.25) was implanted to repair a 10-mm sciatic nerve defect in rats. The results indicated improvements in sciatic functional index, electrophysiology, and sciatic nerve and gastrocnemius muscle histology compared with the CHGFC-0 group, and similar outcomes to the autograft group. In conclusion, we provide a candidate method for the repair of peripheral nerve defects using free-standing chitosan/GO nerve conduits produced by electrodeposition.

Sutureless neurorrhaphy system using a macrophage-polarizing in situ visible light-crosslinkable adhesive protein hydrogel for functional nerve regeneration

Surgical intervention after traumatic peripheral nerve injury relies heavily on sutures. While the suture method often burdens functional nerve regeneration by causing secondary traumatic damage and eliciting prolonged inflammatory immune responses, replacing sutures has rarely been considered because secure anastomosis between severed nerve ends has been the utmost priority in neurorrhaphy. In the present work, a macrophage-polarizing sutureless neurorrhaphy system was proposed by using an in situ visible light-crosslinkable protein-based bioadhesive hydrogel containing a functional neurotransmitter peptide. The efficacy of the proposed bioadhesive hydrogel as a successful sutureless neurorrhaphy system was intensively evaluated in vitro and in vivo. Using a rat sciatic nerve defect model, our findings suggested that this macrophage-polarizing bioadhesive hydrogel offered effective sutureless anastomosis and the capability to induce M2 macrophage polarization for effective tissue remodeling, significantly enhancing functional nerve regeneration compared with conventional sutures. Taken together, these results suggest that our macrophage-polarizing functional bioadhesive hydrogel system can be utilized as a promising alternative to surgical sutures for nerve regenerative medicine applications.

Multifunctional Double-Layer Composite Hydrogel Conduit Based on Chitosan for Peripheral Nerve Repairing.

Peripheral nerve regeneration and functional recovery is a major challenge in clinical practice. Nerve conduit is an effective treatment for peripheral nerve repair, but the traditional hollow nerve conduit is not satisfactory in peripheral nerve repair due to the limitation of cell migration and nutrient transport. Herein, the double cross-linked hydrogels with injectable, self-healing, and conductive properties are synthesized by the Schiff base reaction between polyaniline-modified carboxymethyl chitosan and aldehyde-modified Pluronic F-127 (F127-CHO), and the hydrophobic interaction of F127-CHO. The conductive hydrogel is injected into the cavity of chitosan conduit prepared by electrodeposition. The inner conductive hydrogel and the outer chitosan conduit are formed into a whole through the Schiff base reaction to obtain a double-layer composite hydrogel nerve conduit. The double-layer composite hydrogel neural conduit loaded with 7,8-dihydroxyflavone (DHF) has excellent degradability, biocompatibility, antioxidant activity, and Schwann cell proliferation activity. In the rat sciatic nerve defect model, the double-layer composite hydrogel nerve conduit significantly promotes sciatic nerve regeneration compared with the chitosan hollow conduit. Surprisingly, the repair ability of double-layered hydrogel nerve conduit loaded with DHF is comparable to that of autologous transplantation. Therefore, this multifunctional double-layer composite hydrogel conduit has great potential for peripheral nerve repairing.

A Combined Conduit-Bioactive Hydrogel Approach for Regeneration of Transected Sciatic Nerves.

Transected peripheral nerve injury (PNI) affects the quality of life of patients, which leads to socioeconomic burden. Despite the existence of autografts and commercially available nerve guidance conduits (NGCs), the complexity of peripheral nerve regeneration requires further research in bioengineered NGCs to improve surgical outcomes. In this work, we introduce multidomain peptide (MDP) hydrogels, as intraluminal fillers, into electrospun poly(ε-caprolactone) (PCL) conduits to bridge 10 mm rat sciatic nerve defects. The efficacy of treatment groups was evaluated by electromyography and gait analysis to determine their electrical and motor recovery. We then studied the samples' histomorphometry with immunofluorescence staining and automatic axon counting/measurement software. Comparison with negative control group shows that PCL conduits filled with an anionic MDP may improve functional recovery 16 weeks postoperation, displaying higher amplitude of compound muscle action potential, greater gastrocnemius muscle weight retention, and earlier occurrence of flexion contracture. In contrast, PCL conduits filled with a cationic MDP showed the least degree of myelination and poor functional recovery. This phenomenon may be attributed to MDPs' difference in degradation time. Electrospun PCL conduits filled with an anionic MDP may become an attractive tissue engineering strategy for treating transected PNI when supplemented with other bioactive modifications.

Polydopamine-coated polycaprolactone/carbon nanotube fibrous scaffolds loaded with brain-derived neurotrophic factor for peripheral nerve regeneration

Carbon nanotubes (CNTs) have attracted increasing attention in the field of peripheral nerve tissue engineering due to their unique structural and physical characteristics. In this study, a novel type of aligned conductive scaffolds composed of polycaprolactone (PCL) and CNTs were fabricated via electrospinning. Utilizing mussel-inspired polydopamine (PDA) surface modification, brain-derived neurotrophic factor (BDNF) was loaded onto PCL/CNT fibrous scaffolds to obtain PCL/CNT-PDA-BDNF fibrous scaffolds capable of the sustained release of BDNF over 28 d. Schwann cells were cultured on these scaffolds, and the effect of the scaffolds on peripheral nerve regeneration in vitro was assessed by studying cell proliferation, morphology and the expressions of myelination-related genes S100, P0 and myelin basic protein. Furthermore, the effect of these scaffolds on peripheral nerve regeneration in vivo was investigated using a 10 mm rat sciatic nerve defect model. Both the in vitro and in vivo results indicate that PCL/CNT-PDA-BDNF fibrous scaffolds effectively promote sciatic nerve regeneration and functional recovery. Therefore, PCL/CNT-PDA-BDNF fibrous scaffolds have great potential for peripheral nerve restoration.

Acellular nerve xenografts based on supercritical extraction technology for repairing long-distance sciatic nerve defects in rats.

Compared to conventional artificial nerve guide conduits (NGCs) prepared using natural polymers or synthetic polymers, acellular nerve grafts (ACNGs) derived from natural nerves with eliminated immune components have natural bionic advantages in composition and structure that polymer materials do not have. To further optimize the repair effect of ACNGs, in this study, we used a composite technology based on supercritical carbon dioxide (scCO2) extraction to process the peripheral nerve of a large mammal, the Yorkshire pig, and obtained an innovative Acellular nerve xenografts (ANXs, namely, CD + scCO2 NG). After scCO2 extraction, the fat and DNA content in CD + scCO2 NG has been removed to the greatest extent, which can better supported cell adhesion and proliferation, inducing an extremely weak inflammatory response. Interestingly, the protein in the CD + scCO2 NG was primarily involved in signaling pathways related to axon guidance. Moreover, compared with the pure chemical decellularized nerve graft (CD NG), the DRG axons grew naturally on the CD + scCO2 NG membrane and extended long distances. In vivo studies further revealed that the regenerated nerve axons had basically crossed the CD + scCO2 NG 3 weeks after surgery. 12 weeks after surgery, CD + scCO2 NG was similar to autologous nerves in improving the quality of nerve regeneration, target muscle morphology and motor function recovery and was significantly better than hollow NGCs and CD NG. Therefore, we believe that the fully decellularized and fat-free porcine ACNGs may be the most promising “bridge” for repairing human nerve defects at this stage and for some time to come.

Experimental study on the construction of telmisartan/collagen/polycaprolactone nerve conduit and its repair effect on rat sciatic nerve defect

To construction the telmisartan/collagen/polycaprolactone (PCL) nerve conduit and assess its effect on repairing sciatic nerve defect in rats. The 60% collagen/hexafluoroisopropanol (HFIP) solution and 40% PCL/HFIP solution were prepared and mixed (collagen/PCL solution). Then the 0, 5, 10, and 20 mg of telmisartan were mixed with the 10 mL collagen/PCL solution, respectively. Telmisartan/collagen/PCL nerve conduits were fabricated via high voltage electrospinning technology. The structure of nerve conduit before and after crosslinking was observed by using scanning electron microscope (SEM). The drug release efficiency was detected by in vitro sustained release method. RAW264.7 cells were cultured with lipopolysaccharide to induce inflammation, and then co-cultured with nerve conduits loaded with different concentrations of telmisartan for 24 hours. The mRNA expressions of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) were detected by using real-time fluorescence quantitative PCR. Forty adult Wistar rats were randomly divided into 4 groups ( n=10). After preparing 15-mm-long sciatic nerve defect, the defect was repaired by cross-linked nerve conduits loaded with 0, 5, 10, and 20 mg telmisartan in groups A, B, C, and D, respectively. After operation, the general condition of rats was observed after operation; the sciatic function index (SFI) was tested; the bridging between the nerve conduit and sciatic nerve, and the integrity of nerve conduit were observed; the tissue growth in nerve conduit and material degradation were observed by HE staining; the expressions of CD86 (M1 macrophage marker), CD206 (M2 macrophage marker), myelin basic protein (MBP), and myelin protein 0 (P0) in new tissues were also observed by immunohistochemical staining; the expressions of neurofilament 200 (NF-200) and S-100β in new tissues were assessed by immunofluorescence staining. The general observation showed that the inner diameter of the nerve conduit was 1.8 mm and the outer diameter was 2.0 mm. After cross-linking by genipin, the nanofiber became thicker and denser. The drug release test showed that the telmisartan loaded nerve conduit could be released gradually. With the increase of telmisartan content in nerve conduit, the iNOS mRNA expression decreased and the Arg-1 mRNA expression increased; and the differences between 20 mg group and other groups were significant ( P<0.05). In vivo experiment showed that all animals in each group survived until the completion of the experiment. The SFI was significantly higher in groups C and D than in groups A and B at different time points ( P<0.05) and in group D than in group C at 6 months after operation ( P<0.05). HE staining showed that there were significantly more new tissues in the middle of the nerve conduit in group D after operation than in other groups. Immunohistochemical staining showed that CD86 and CD206 stainings were positive in each group at 1 month after operation, among which group D had the lowest positive rate of CD86 and the highest positive rate of CD206, and there were significant differences in the positive rate of CD206 between group D and groups A, B, and C ( P<0.05); the MBP and P0 stainings were positive in groups C and D at 6 months, and the positive rate in group D was significantly higher than that in group C ( P<0.05). Immunofluorescence staining showed that the NF-200 and S-100β expressions in group D were significantly higher than those in other groups. Telmisartan/collagen/PLC nerve conduit can promote the sciatic nerve defect repair in rats through promoting the polarization of M1 macrophages to M2 macrophages, and the nerve conduit loaded with20 mg telmisartan has the most significant effect.

Conductive Nerve Guidance Conduits Based on Morpho Butterfly Wings for Peripheral Nerve Repair.

Peripheral nerve injury (PNI), causing loss of sensory and motor function, is a complex and challenging disease in the clinic due to the restricted regeneration capacity. Nerve guidance conduits (NGCs) have become a promising substitute for peripheral nerve regeneration, but their efficacy is often limited. Here, inspired by the physiological structures of peripheral nerves, we present a conductive topological scaffold for nerve repair by modifying Morpho butterfly wing with reduced graphene oxide (rGO) nanosheets and methacrylated gelatin (GelMA) hydrogel encapsulated brain-derived neurotrophic factor (BDNF). Benefiting from the biocompatibility of GelMA hydrogel, the conductivity of rGO and parallel nanoridge structures of wing scales, PC12 cells, and neural stem cells grown on the modified wing have an increased neurite length with guided cellular orientation. In addition, the NGCs are successfully obtained by manually rolling up the scaffolds and exhibited great performance in repairing 10 mm sciatic nerve defects in rats, and we believe that the NGCs can be applied in reparing longer nerve defects in the future by further optimization. We also demonstrate the feasibility of electrically conductive NGCs based on the rGO/BDNF/GelMA-integrated Morpho butterfly wing as functional nerve regeneration conduits, which may have potential value for application in repairing peripheral nerve injuries.

Sustainable release of nerve growth factor for peripheral nerve regeneration using nerve conduits laden with Bioconjugated hyaluronic acid-chitosan hydrogel

Peripheral nerve regeneration following injury is often incomplete, resulting in significant personal and socioeconomic costs. Nerve conduits have been developed to complement or replace autologous nerve grafts. In this study, repairing injured peripheral nerves in rats is investigated by fabricating poly(D, l-lactic acid) (PDLLA)/β-tricalcium phosphate (β-TCP) nerve conduits filled by injectable chitosan (CS)-hyaluronic acid (HA) hydrogels featuring sustained release of nerve growth factor (NGF). All CS-HA hydrogels have porosities higher than 80%. The mass loss of the CS-HA hydrogels reaches 70% in 8 weeks. In vitro experiments suggest that the CS-HA/NGF hydrogels are suitable for the adhesion, spreading, and differentiation of neuronal cells, which also allow for sustained release of NGF. In vivo studies on the repair of 10-mm sciatic nerve defects in rats are used to assess nerve repair rate through walking track analysis, gastrocnemius weight analysis, and histological analysis. It is shown that the PDLLA/β-TCP nerve conduits laden with CS-HA/NGF hydrogels have a significant positive effect on the axon regeneration and myelination, compared to the PDLLA/β-TCP hollow nerve conduits and the autograft group. These results show that the CS-HA/NGF injectable hydrogel can effectively promote nerve regeneration making it a good candidate in the field of neural tissue engineering.

Development of a regenerative porous PLCL nerve guidance conduit with swellable hydrogel-based microgrooved surface pattern via 3D printing

Peripheral nerve injury causes severe loss of motor and sensory functions, consequently increasing morbidity in affected patients. An autogenous nerve graft is considered the current gold standard for reconstructing nerve defects and recovering lost neurological functions; however, there are certain limitations to this method, such as limited donor nerve supply. With advances in regenerative medicine, recent research has focused on the fabrication of tissue-engineered nerve grafts as promising alternatives to the autogenous nerve grafts. In this study, we designed a nerve guidance conduit using an electrospun poly(lactide-co-ε-caprolactone) (PLCL) membrane with a visible light-crosslinked gelatin hydrogel. The PLCL nanoporous membrane with permeability served as a flexible and non-collapsible epineurium for the nerve conduit; the inner-aligned gelatin hydrogel paths were fabricated via 3D printing and a photocrosslinking system. The resultant gelatin hydrogel with microgrooved surface pattern was established as a conducting guidance path for the effective regeneration of axons and served as a reservoir that can incorporate and release bioactive molecules. From in vivo performance tests using a rat sciatic nerve defect model, our PLCL/gelatin conduit demonstrated successful axonal regeneration, remyelination capacities and facilitated functional recovery. Hence, the PLCL/gelatin conduit developed in this study is a promising substitute for autogenous nerve grafts. Statement of significanceNerve guidance conduits (NGCs) are developed as promising recovery techniques for bridging peripheral nerve defects. However, there are still technological limitations including differences in the structures and components between natural peripheral nerve and NGCs. In this study, we designed a NGC composed of an electrospun poly(lactide-co-ε-caprolactone) (PLCL) membrane and 3D printed inner gelatin hydrogel to serve as a flexible and non-collapsible epineurium and a conducting guidance path, respectively, to mimic the fascicular structure of the peripheral nerve. In particular, in vitro cell tests clearly showed that gelatin hydrogel could guide the cells and function as a reservoir that incorporate and release nerve growth factor. From in vivo performance tests, our regenerative conduit successfully led to axonal regeneration with effective functional recovery.

Multi-factorial nerve guidance conduit engineering improves outcomes in inflammation, angiogenesis and large defect nerve repair

Nerve guidance conduits (NGCs) are sub-optimal for long-distance injuries with inflammation and poor vascularization related to poor axonal repair. This study used a multi-factorial approach to create an optimized biomaterial NGC to address each of these issues. Through stepwise optimization, a collagen-chondroitin-6-sulfate (Coll-CS) biomaterial was functionalized with extracellular matrix (ECM) components; fibronectin, laminin 1 and laminin 2 (FibL1L2) in specific ratios. A snap-cooled freeze-drying process was then developed with optimal pore architecture and alignment to guide axonal bridging. Culture of adult rat dorsal root ganglia on NGCs demonstrated significant improvements in inflammation, neurogenesis and angiogenesis in the specific Fib:L1:L2 ratio of 1:4:1. In clinically relevant, large 15 mm rat sciatic nerve defects, FibL1L2-NGCs demonstrated significant improvements in axonal density and angiogenesis compared to unmodified NGCs with functional equivalence to autografts. Therefore, a multiparameter ECM-driven strategy can significantly improve axonal repair across large defects, without exogenous cells or growth factors.

OP7: Silk Fibroin-Based Conduits Filled with Native Spider Silk Fibers Successfully Promoted Nerve Regeneration in a 10 Mm Sciatic Nerve Defect in Rats

OP7: Silk Fibroin-Based Conduits Filled with Native Spider Silk Fibers Successfully Promoted Nerve Regeneration in a 10 Mm Sciatic Nerve Defect in Rats

Sustained release of exosomes loaded into polydopamine-modified chitin conduits promotes peripheral nerve regeneration in rats.

Exosomes derived from mesenchymal stem cells are of therapeutic interest because of their important role in intracellular communication and biological regulation. On the basis of previously studied nerve conduits, we designed a polydopamine-modified chitin conduit loaded with mesenchymal stem cell-derived exosomes that release the exosomes in a sustained and stable manner. In vitro experiments revealed that rat mesenchymal stem cell-derived exosomes enhanced Schwann cell proliferation and secretion of neurotrophic and growth factors, increased the expression of Jun and Sox2 genes, decreased the expression of Mbp and Krox20 genes in Schwann cells, and reprogrammed Schwann cells to a repair phenotype. Furthermore, mesenchymal stem cell-derived exosomes promoted neurite growth of dorsal root ganglia. The polydopamine-modified chitin conduits loaded with mesenchymal stem cell-derived exosomes were used to bridge 2 mm rat sciatic nerve defects. Sustained release of exosomes greatly accelerated nerve healing and improved nerve function. These findings confirm that sustained release of mesenchymal stem cell-derived exosomes loaded into polydopamine-modified chitin conduits promotes the functional recovery of injured peripheral nerves.

The efficacy of combining a vascularized biogenic conduit and a decellularized nerve graft in the treatment of peripheral nerve defects: An experimental study using the rat sciatic nerve defect model.

Although decellularized nerve grafts are often used as a bridging material in nerve defect repair, the lack of perfusion support in this procedure limits the regeneration capacity. To address this, we applied vascularized biogenic conduits, which are fibrous membranes prefabricated around the silicone rod that contain rich vascularity and macrophages, to nerve defect repair procedures using decellularized nerve grafts. The purpose of this study is to investigate the capacity of combining a vascularized biogenic conduit and a decellularized nerve graft for peripheral nerve regeneration using a 10-mm nerve defect model in rats. Sixteen adult male rats (F344 rats, 10-12 weeks, 200-250 g) were used in this study. For the prefabrication of vascularized biogenic conduits, a silicone rod was transplanted next to the sciatic nerve. After 8 weeks, this silicone rod was enveloped in connective tissue, called a vascularized biogenic conduit. The first rat was used to investigate the histological characteristics of vascularized biogenic conduits through immunofluorescence studies. The remaining 15 rats were divided into three groups to evaluate the efficacy of the combination of a decellularized nerve graft and a vascularized biogenic conduit: a decellularized nerve graft (DNG) group, a decellularized nerve graft with a vascularized biogenic conduit (DNG-w-VBC) group, and an autologous nerve graft (ANG) group. Eight weeks after nerve graft surgery, the assessment results of both functional recovery (electrophysical studies and target muscle atrophy) and morphological recovery (total number, diameter, and myelin thickness of the regenerated axons) of the regenerated nerves were examined. Immunofluorescence studies revealed that the VBC contains extracellular matrix, vascular tissue, and macrophages. The results of the DNG-w-VBC group were superior to the DNG group in electrophysiological studies (CMAP; 6.29 ± 0.80% vs. 4.02 ± 3.35%, MNCV; 50.6 ± 8.4% vs. 25.7 ± 15.6%, p < .05, respectively), regenerated axon number (11,348 ± 812 vs. 7697 ± 2197, p < .05), and mean axon diameter (2.72 ± 0.33 μm vs. 1.64 ± 0.12 μm, p < .05). Our study confirms that vascularized biogenic conduits supply vascularity and macrophages to nerve defect sites. Combining vascularized biogenic conduits with decellularized nerve grafts to treat nerve defects offers superior functional and morphological recovery of regenerated axons.

Recombinant COL6 α2 as a Self-Organization Factor That Triggers Orderly Nerve Regeneration Without Guidance Cues.

Collagen VI (COL6) in the microenvironment was recently identified as an extracellular signal that bears the function of promoting orderly axon bundle formation. However, the large molecular weight of COL6 (≈2,000 kDa) limits its production and clinical application. It remains unclear whether the smaller subunit α chains of COL6 can exert axon bundling and ordering effects independently. Herein, based on a dorsal root ganglion (DRG) ex vivo model, the contributions of three main COL6 α chains on orderly nerve bundle formation were analyzed, and COL6 α2 showed the largest contribution weight. A recombinant COL6 α2 chain was produced and demonstrated to promote the formation of orderly axon bundles through the NCAM1-mediated pathway. The addition of COL6 α2 in conventional hydrogel triggered orderly nerve regeneration in a rat sciatic nerve defect model. Immunogenicity assessment showed weaker immunogenicity of COL6 α2 compared to that of the COL6 complex. These findings suggest that recombinant COL6 α2 is a promising material for orderly nerve regeneration.

A nerve conduit filled with Wnt5a-loaded fibrin hydrogels promotes peripheral nerve regeneration.

AimsPeripheral nerve injury is a significant clinical problem with a substantial impact on quality of life, for which no optimal treatment has been found. This study aimed to analyze the effect and mechanism of Wnt5a‐loaded fibrin hydrogel on a 10‐mm rat sciatic nerve defect.MethodsThe Wnt5a‐loaded fibrin hydrogel was synthesized by mixing a Wnt5a solution with thrombin and fibrinogen solutions. The loading capacity and release profile of Wnt5a‐loaded fibrin hydrogel and the effect of Wnt5a on Schwann cells were evaluated in vitro. We also assessed the in vivo repair status via histological analysis of the regenerative nerve and gastrocnemius muscle, electrophysiological examination, gait analysis, and muscle wet weight.ResultsWe developed a nerve conduit filled with Wnt5a‐loaded fibrin hydrogel (Fn) as a sustained‐release system to repair a 10‐mm rat sciatic nerve defect. In vitro, Wnt5a could promote SC proliferation and the gene expression of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and cholinergic neurotrophic factor (CNTF), as well as the protein secretion of VEGF and NGF. In vivo, the Wnt5a/Fn group was superior to the hollow, fibrin hydrogel, and Wnt5a groups in terms of axonal growth, myelination, electrophysiological recovery, target organ innervation, and motor function recovery 12 weeks after the operation.ConclusionThe Wnt5a/Fn nerve conduit can promote peripheral nerve defect regeneration, with potential clinical applications. The mechanism for this may be the facilitation of multiple neurotrophin secretion, combining vascularization and neurotrophic growth cues.

In Situ Fabrication of Nerve Growth Factor Encapsulated Chitosan Nanoparticles in Oxidized Bacterial Nanocellulose for Rat Sciatic Nerve Regeneration.

Autograft is currently the gold standard in the clinical treatment of peripheral nerve injury (PNI), which, however, is limited by the availability of a donor nerve and secondary injuries. Nerve guidance conduits (NGC) provide a suitable microenvironment to promote the regeneration of injured nerves, which could be the substitutes for autografts. In this study, nerve growth factor (NGF) encapsulated chitosan nanoparticles (CSNPs) were first constructed in situ in an oxidized bacterial cellulose (OBC) conduit using the ion gel method after the introduction of a CS/NGF solution under pressure to enable a sustainable release of NGF. A novel NGF@CSNPs/OBC nanocomposite with antibacterial activity, biodegradability, and porous microstructure was successfully developed. In vitro experiments showed that the nanocomposite promoted the adhesion and proliferation of Schwann cells. When the nanocomposite was applied as NGC to repair the sciatic nerve defect of rats, a successful repair of the 10 mm nerve defect was observed after 4 weeks. At week 9, the diameter, morphology, histology, and functional recovery of the regenerated nerve was comparable to the autografts, indicating that the NGC effectively promoted the regeneration and function recovery of the nerve. In summary, the NGF@CSNPs/OBC as a novel NGC provides great potential in the treatment of PNI.

In Situ Prevascularization Strategy with Three-Dimensional Porous Conduits for Neural Tissue Engineering.

Neovascularization is crucial for peripheral nerve regeneration and long-term functional restoration. Previous studies have emphasized strategies that enhance axonal repair over vascularization. Here, we describe the development and application of an in situ prevascularization strategy that uses 3D porous nerve guidance conduits (NGCs) to achieve angiogenesis-mediated neural regeneration. The optimal porosity of the NGC is a critical feature for achieving neovascularization and nerve growth patency. Hollow silk fibroin/poly(l-lactic acid-co-ε-caprolactone) NGCs with 3D sponge-like walls were fabricated using electrospinning and freeze-drying. In vitro results showed that 3D porous NGC favored cell biocompatibility had neuroregeneration potential and, most importantly, had angiogenic activity. Results from our mechanistic studies suggest that activation of HIF-1α signaling might be associated with this process. We also tested in situ prevascularized 3D porous NGCs in vivo by transplanting them into a 10 mm rat sciatic nerve defect model with the aim of regenerating the severed nerve. The prevascularized 3D porous NGCs greatly enhanced intraneural angiogenesis, resulting in demonstrable neurogenesis. Eight weeks after transplantation, the performance of the prevascularized 3D NGCs was similar to that of traditional autografts in terms of improved anatomical structure, morphology, and neural function. In conclusion, combining a reasonably fabricated 3D-pore conduit structure with in situ prevascularization promoted functional nerve regeneration, suggesting an alternative strategy for achieving functional recovery after peripheral nerve trauma.