Urethral Microenvironment Adapted Sodium Alginate/Gelatin/Reduced Graphene Oxide Biomimetic Patch Improves Scarless Urethral Regeneration.

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 Ca2+ 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.

Fabrication of SA/Gel/C scaffold with 3D bioprinting to generate micro-nano porosity structure for skin wound healing: a detailed animal in vivo study.

Bioprinting has exhibited remarkable promises for the fabrication of functional skin substitutes. However, there are some significant challenges for the treatment of full-thickness skin defects in clinical practice. It is necessary to determine bioinks with suitable mechanical properties and desirable biocompatibilities. Additionally, the key for printing skin is to design the skin structure optimally, enabling the function of the skin. In this study, the full-thickness skin scaffolds were prepared with a gradient pore structure constructing the dense layer, epidermis, and dermis by different ratios of bioinks. We hypothesized that the dense layer protects the wound surface and maintains a moist environment on the wound surface. By developing a suitable hydrogel bioink formulation (sodium alginate/gelatin/collagen), to simulate the physiological structure of the skin via 3D printing, the proportion of hydrogels was optimized corresponding to each layer. These results reveal that the scaffold has interconnected macroscopic channels, and sodium alginate/gelatin/collagen scaffolds accelerated wound healing, reduced skin wound contraction, and re-epithelialization in vivo. It is expected to provide a rapid and economical production method of skin scaffolds for future clinical applications.

Modulation of myelin formation by combined high affinity with extracellular matrix structure of electrospun silk fibroin nanoscaffolds.

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.

Novel conductive polypyrrole/silk fibroin scaffold for neural tissue repair.

Three dimensional (3D) bioprinting, which involves depositing bioinks (mixed biomaterials) layer by layer to form computer-aided designs, is an ideal method for fabricating complex 3D biological structures. However, it remains challenging to prepare biomaterials with micro-nanostructures that accurately mimic the nanostructural features of natural tissues. A novel nanotechnological tool, electrospinning, permits the processing and modification of proper nanoscale biomaterials to enhance neural cell adhesion, migration, proliferation, differentiation, and subsequent nerve regeneration. The composite scaffold was prepared by combining 3D bioprinting with subsequent electrochemical deposition of polypyrrole and electrospinning of silk fibroin to form a composite polypyrrole/silk fibroin scaffold. Fourier transform infrared spectroscopy was used to analyze scaffold composition. The surface morphology of the scaffold was observed by light microscopy and scanning electron microscopy. A digital multimeter was used to measure the resistivity of prepared scaffolds. Light microscopy was applied to observe the surface morphology of scaffolds immersed in water or Dulbecco's Modified Eagle's Medium at 37°C for 30 days to assess stability. Results showed characteristic peaks of polypyrrole and silk fibroin in the synthesized conductive polypyrrole/silk fibroin scaffold, as well as the structure of the electrospun nanofiber layer on the surface. The electrical conductivity was 1 × 10-5-1 × 10-3 S/cm, while stability was 66.67%. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was employed to measure scaffold cytotoxicity in vitro. Fluorescence microscopy was used to observe EdU-labeled Schwann cells to quantify cell proliferation. Immunohistochemistry was utilized to detect S100ß immunoreactivity, while scanning electron microscopy was applied to observe the morphology of adherent Schwann cells. Results demonstrated that the polypyrrole/silk fibroin scaffold was not cytotoxic and did not affect Schwann cell proliferation. Moreover, filopodia formed on the scaffold and Schwann cells were regularly arranged. Our findings verified that the composite polypyrrole/silk fibroin scaffold has good biocompatibility and may be a suitable material for neural tissue engineering.

Genipin-Cross-Linked Chitosan Nerve Conduits Containing TNF-α Inhibitors for Peripheral Nerve Repair.

Tissue engineered nerve grafts (TENGs) are considered a promising alternative to autologous nerve grafting, which is considered the "gold standard" clinical strategy for peripheral nerve repair. Here, we immobilized tumor necrosis factor-α (TNF-α) inhibitors onto a nerve conduit, which was introduced into a chitosan (CS) matrix scaffold utilizing genipin (GP) as the crosslinking agent, to fabricate CS-GP-TNF-α inhibitor nerve conduits. The in vitro release kinetics of TNF-α inhibitors from the CS-GP-TNF-α inhibitor nerve conduits were investigated using high-performance liquid chromatography. The in vivo continuous release profile of the TNF-α inhibitors released from the CS-GP-TNF-α inhibitor nerve conduits was measured using an enzyme-linked immunosorbent assay over 14 days. We found that the amount of TNF-α inhibitors released decreased with time after the bridging of the sciatic nerve defects in rats. Moreover, 4 and 12 weeks after surgery, histological analyses and functional evaluations were carried out to assess the influence of the TENG on regeneration. Immunochemistry performed 4 weeks after grafting to assess early regeneration outcomes revealed that the TENG strikingly promoted axonal outgrowth. Twelve weeks after grafting, the TENG accelerated myelin sheath formation, as well as functional restoration. In general, the regenerative outcomes following TENG more closely paralleled findings observed with autologous grafting than the use of the CS matrix scaffold. Collectively, our data indicate that the CS-GP-TNF-α inhibitor nerve conduits comprised an elaborate system for sustained release of TNF-α inhibitors in vitro, while studies in vivo demonstrated that the TENG could accelerate regenerating axonal outgrowth and functional restoration. The introduction of CS-GP-TNF-α-inhibitor nerve conduits into a scaffold may contribute to an efficient and adaptive immune microenvironment that can be used to facilitate peripheral nerve repair.

Construction of polyacrylamide/graphene oxide/gelatin/sodium alginate composite hydrogel with bioactivity for promoting Schwann cells growth.

Various hydrogels made from natural or synthetic polymers have been widely used in biologic tissues, drug delivery, and artificial implants due to their good biocompatibility, indicating a promising perspective in regenerative medicine. In the present study, a composite hydrogel named polyacrylamide/graphene oxide/gelatin/sodium alginate (PAM/GO/Gel/SA) for accelerating peripheral nerve regeneration was fabricated through in situ free radical polymerization for the first time. A series of physicochemical properties including morphology, porosity, swelling behaviors, component, mechanical properties, and in vitro degradation behavior of the prepared composite hydrogel were characterized. The effects of the composite hydrogel on Schwann cells growth were evaluated and the related molecular mechanism was further penetrated. The results showed that the prepared PAM/GO/Gel/SA composite hydrogels displayed different color appearance as the function of component variations. The surface morphology, components, swelling ratio, mechanical properties, and porosity were all changed with the concentration alteration of each ingredient, while no obvious degradation behavior was observed, indicating a controllable physicochemical property. The culture of cells exhibited that the composite hydrogels could well support the attachment and proliferation of Schwann cells. The gene expression levels of Sox10, GAP43, and myelin basic protein (MBP) in PGG0.5 SYR1 and PGG1 SYR0.5 were higher than those of NC. This study may provide important theoretical and experimental basis for the design and development of hydrogel scaffolds for nerve tissue engineering application. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1951-1964, 2018.

A new electrospun graphene-silk fibroin composite scaffolds for guiding Schwann cells.

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.

Chitosan degradation products facilitate peripheral nerve regeneration by improving macrophage-constructed microenvironments.

Chitosan-based artificial nerve grafts have been widely employed to repair peripheral nerve defects. Our previous study has shown that chitosan constructed nerve graft not only provides suitable scaffolds for nerve regeneration, its degradation products, chitooligosaccharides (COS), also promote nerve repair. However, the involved mechanisms are still not fully elucidated. In the present study, we observed that pro-inflammatory cytokines, as well as macrophage infiltration, were transiently up-regulated in the injured sciatic nerves which were bridged with silicon tubes filled with COS. Based upon transcriptome analysis, the axis of miR-327/CCL2 in Schwann cells (SCs) was identified as a potential target of COS. The following experiments have confirmed that COS stimulate CCL2 expression by down-regulating miR-327 in SCs. Consequently, the resulting CCL2 induces macrophage migration at injury sites to re-construct microenvironments and thus facilitates nerve regeneration. Collectively, our data provide a theoretical basis for the clinical application of chitosan-based grafts in peripheral nerve regeneration.

[Construction and biocompatibility in vitro evaluation of electrospun-graphene/silk fibroin nanofilms].

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.