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The nasty urine microenvironment (UME) is an inherent obstacle that hinders urethral repair due to fibrosis and swelling of the oftentimes adopted hydrogel-based biomaterials. Here, using reduced graphene oxide (rGO) along with double-freeze-drying to strengthen a 3D-printed patch is reported to realize scarless urethral repair. The sodium alginate/gelatin/reduced graphene oxide (SA/Gel/rGO) biomaterial features tunable stiffness, degradation profile, and anti-fibrosis performance. Interestingly, the 3D-printed alginate-containing composite scaffold is able to respond to Ca 2+ present in the urine, leading to enhanced structural stability and strength as well as inhibiting swelling. The investigations present that the swelling behaviors, mechanical properties, and anti-fibrosis efficacy of the SA/Gel/rGO patch can be modulated by varying the concentration of rGO. In particular, rGO in optimal concentration shows excellent cell viability, migration, and proliferation. In-depth mechanistic studies reveal that the activation of cell proliferation and angiogenesis-related proteins, along with inhibition of fibrosis-related gene expressions, play an important role in scarless repair by the 3D-printed SA/Gel/rGO patch via promoting urothelium growth, accelerating angiogenesis, and minimizing fibrosis in vivo. The proposed strategy has the potential of resolving the dilemma of necessary biomaterial stiffness and unwanted fibrosis in urethral repair., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Remyelination is a major therapeutic goal in peripheral nerve regeneration, serving to restore function of demyelinated axons and provide neuroprotection. In order to apply myelin biogenesis strategies to peripheral nerve defects, the tissue engineered substitutes might be amenable to the promotion of this repair process. Electrospun nanofibers are considered as promising scaffolds for tissue engineering due to extracellular matrix mimicking factor and enhanced electrostatic interaction resulting in a controllable 3 D nanofibrous membrane. In order to explore the role of electrospun silk fibroin (SF) membrane in myelination, co-culture of dorsal root ganglion (DRG) neurons and Schwann cells (SCs) in vitro was established and observed. Scanning electron microscopy was used to observe DRG adhesion to the membranes, the electrospinning SF membrane is more favorable to the adhesion of DRG. The immunofluorescence staining of MAG and NF showed considerable amount of myelin were formed, and the myelin was tightly wrapped around the axons of the neurons, which was confirmed under the scanning electron microscope observation. Real-time quantitative PCR technique was used to determine the gene expression level of DRG neurons cultured at different time points. The results showed that the mRNA levels of N-cadherin, laminin, fibronectin were higher than those in the control group. Our results showed that the electrospun SF nanofibers can provide topographical and chemical cues that mimic (to a certain extent) the extracellular matrix.

Graphene (Gr) has been made of various forms used for repairing peripheral nerve injury with favorable electroactivity, however, graphene-based scaffolds in peripheral nerve regeneration are still rarely reported due to the difficulty of realizing uniform dispersion of graphene and electroactive materials at nanoscale as well as lacking biocompatibility. In this paper, graphene-silk fibroin (SF) composite nanofiber membranes with different mass ratios were prepared via electrospinning. Microscopic observation revealed that electrospun Gr/SF membranes had a nanofibrous structure. Electrochemical analysis provided electroactivity characterization of the Gr/SF membranes. The physiochemical results showed that the physiochemical properties of electrospun Gr/SF membranes could be changed by varying Gr concentration. Swelling ratio and contact angle measurements confirmed that electrospun Gr/SF membranes possessed large absorption capacity and hydrophilic surface, and the mechanical property was improved with increasing Gr concentration. Additionally, in-vitro cytotoxicity with L929 revealed that all the electrospun Gr/SF membranes are biocompatible. Moreover, the morphology and quantity showed that the membranes supported the survival and growth of the cultured Schwann cells. Collectively, all of the results suggest that the electrospun Gr/SF membranes combine the excellent electrically conductivity and mechanical strength of the graphene with biocompatibility property of silk to mimic the natural neural cell micro-environment for nerve development.

Objective: To explore the construction and biocompatibility in vitro evaluation of the electrospun-graphene (Gr)/silk fibroin (SF) nanofilms., Methods: The electrostatic spinning solution was prepared by dissolving SF and different mass ratio (0, 5%, 10%, 15%, and 20%) of Gr in formic acid solution. The hydrophilia and hydrophobic was analyzed by testing the static contact angle of electrostatic spinning solution of different mass ratio of Gr. Gr-SF nanofilms with different mass ratio (0, 5%, 10%, 15%, and 20%, as groups A, B, C, D, and E, respectively) were constructed by electrospinning technology. The structure of nanofilms were observed by optical microscope and scanning electron microscope; electrochemical performance of nanofilms were detected by cyclic voltammetry at electrochemical workstation; the porosity of nanofilms were measured by n-hexane substitution method, and the permeability were observed; L929 cells were used to evaluate the cytotoxicity of nanofilms in vitro at 1, 4, and 7 days after culture. The primary Sprague Dawley rats' Schwann cells were co-cultured with different Gr-SF nanofilms of 5 groups for 3 days, the morphology and distribution of Schwann cells were identified by toluidine blue staining, the cell adhesion of Schwann cells were determined by cell counting kit 8 (CCK-8) method, the proliferation of Schwann cells were detected by EdU/Hoechst33342 staining., Results: The static contact angle measurement confirmed that the hydrophilia of Gr-SF electrospinning solution was decreased by increasing the mass ratio of Gr. Light microscope and scanning electron microscopy showed that Gr-SF nanofilms had nanofiber structure, Gr particles could be dispersed uniformly in the membrane, and the increasing of mass ratio of Gr could lead to the aggregation of particles. The porosity measurement showed that the Gr-SF nanofilms had high porosity (>65%). With the increasing of mass ratio of Gr, the porosity and conductivity of Gr-SF nanofilm increased gradually, the value in the group A was significantly lower than those in groups C, D, and E ( P <0.05). In vitro L929 cells cytotoxicity test showed that all the Gr-SF nanofilms had good biocompatibility. Toluidine blue staining, CCK-8 assay, and EdU/Hoechst33342 staining showed that Gr-SF nanofilms with mass ratio of Gr less than 10% could support the survival and proliferation of co-cultured Schwann cells., Conclusion: The Gr-SF nanofilm with mass ratio of Gr less than 10% have proper hydrophilia, conductivity, porosity, and other physical and chemical properties, and have good biocompatibility in vitro . They can be used in tissue engineered nerve preparation.