Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect.

Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide conduits in the field of nerve tissue engineering.

Natural Flammulina velutipes-Based Nerve Guidance Conduit as a Potential Biomaterial for Peripheral Nerve Regeneration: In Vitro and In Vivo Studies.

The treatment and repair of serious peripheral nerve injuries remain challenging in the clinical practice, while the application of multifunctional nerve guidance conduits (NGCs) based on naturally derived polymers has attracted much attention in recent years because of their excellent physicochemical properties and biological characteristics. Flammulina velutipes (Curt. ex FV) is a popular edible mushroom characterized by hollow tubular structures, antibacterial activities, and high nutritional properties. In this study, FV is utilized to construct NGCs (labeled FVC) via a freeze-drying technique without chemical modifications. The morphology, physical properties, cellular biocompatibility, antibacterial properties, and nerve regeneration capacity of FVC were assessed both in vitro and in vivo. FVC is composed of hollow tubes and evenly irregular interconnected micropores with 73.8 ± 5.5% porosity and 476.1 ± 12.9 µm hollow tube diameter. The inner surface of the FVC presents multiple microgrooves elongated parallel to the long axis. Moreover, FVC possessed strong antibacterial activity and could inhibit Gram-positive Staphylococcus aureus growth by up to 96.0% and Gram-negative Escherichia coli growth by up to 94.8% in vitro. FVC exhibited excellent biocompatibility and effectively promoted PC-12 cell proliferation and elongation in vitro. When applied to repair critical-sized sciatic nerve defects, FVC could effectively stimulate nerve functional recovery and axonal outgrowth in a rat model. Interestingly, Western blot analysis indicated that growth-associated protein 43 (GAP-43) had increased expression levels in the FVC group compared with the autograft group. This result suggested that by activating the Janus activated kinase2 (JAK2)/Phosphorylation ofsignal transducer and activator of transcription-3 (STAT3) signaling pathway, FVC upregulated Phosphorylation of signal transducer and activator of transcription-3 (P-STAT3) in vivo, resulting in the secretion of GAP-43. Collectively, a natural NGC FVC was fabricated based on FV without chemical modifications. The morphology, physical properties, cellular biocompatibility, antibacterial properties, and nerve regeneration capacity of FVC provide new insights for its further optimization and application in the field of nerve tissue engineering.

Green fabrication of seedbed-like Flammulina velutipes polysaccharides-derived scaffolds accelerating full-thickness skin wound healing accompanied by hair follicle regeneration.

A novel seedbed-like scaffold was firstly fabricated by the "frozen sectioning" processing method using Flammulina velutipes as a raw material. The Flammulina velutipes polysaccharides scaffold is composed of a natural structure imitating the "ground" (connected and aligned hollow tubes with porous walls). Meanwhile, its biologically active components include polysaccharides and proteins, mimicking the "plant nutrition" in the seedbed. To further optimize the ground and nutrition components, Flammulina velutipes polysaccharides-derived scaffolds (FPDSs) were fabricated via the treatment of original Flammulina velutipes polysaccharides scaffold (labeled FPS) by NaOH, cysteine (labeled as FPS/NaOH, FPS/Cys, respectively). FPDSs were characterized by SEM, FTIR, XRD, water absorption and retention, and mechanical evaluations. From the results, FPS/NaOH and FPS/Cys lost the characteristic big tubes of original strips and had higher water absorption capacities comparing to FPS. Simultaneously, FPS/NaOH had better ductility, FPS/Cys had showed increased stiffness. Biological activities of FPDSs were tested against different types of bacteria exhibiting excellent anti-bacterial activity, and FPS/NaOH and FPS/Cys had dramatically higher anti-bacterial activity than FPS. The cytocompatibility of FPDSs was evaluated utilizing mouse fibroblast cell line (L929), and all FPDSs showed good cytocompatibility. The FPDSs were further applied to a rat full-thickness skin wound model, and they all exhibited obviously accelerated re-epithelialization, among which FPS/NaOH showed the greatest efficiency. FPS/NaOH could shorten the wound-healing process as evidenced by dynamic alterations of the expression levels of specific stagewise markers in the healing areas. Similarly, FPS/NaOH can efficiently induce hair follicle regeneration in the healing skin tissues. In summary, FPDSs exhibit potential functions as seedbeds to promote the regeneration of the "seed" including hair follicles and injured skin, opening a new avenue for wound healing.

Aligned soy protein isolate-modified poly(L-lactic acid) nanofibrous conduits enhanced peripheral nerve regeneration.

OBJECTIVE: Repair and regeneration of peripheral nerve defect by engineered conduits have greatly advanced in the past decades while still facing great challenges. APPROACH: In this work, we fabricated a new highly oriented poly(L-lactic acid) (PLLA)/soy protein isolate (SPI) nanofibrous conduit (HO-PSNC) for nerve regeneration. MAIN RESULTS: Firstly, we observed that SPI could efficiently modify PLLA for the electrospinning of PLLA/SPI nanofibers with enhanced physical and biological properties. Incorporation of SPI decreased the fiber diameter and ductility of PLLA/SPI nanofibrous films (PSNFs), improved the tensile strength and surface wettability of PSNFs and increased the in vivo degradability of the PSNFs. When the hybrid ratio of SPI was 20 and 40%, PSNFs could efficiently promote neural cell extension and differentiation in vitro. Based on these data, 20% SPI (PSNF-20) was chosen for further investigation. Next, PSNF-20 with different fiber orientations (random/low orientation, medium, and high orientation, respectively) were developed and used for evaluating neural cell behaviors on the materials. Results revealed that the PSNF-20 with highly oriented nanofibers (HO-PSNF-20) or mediumly oriented nanofibers (MO-PSNF-20) showed a better performance in directing cell extension and enhancing neurite outgrowth. Finally, the highly oriented nanofibers conduits (HO-PSNC-20) were used to bridge sciatic nerve defect in rats with highly oriented PLLA and autografts as controls. HO-PSNC-20 exhibited a significant promotion in nerve regeneration and functional reconstruction comparing to highly oriented PLLA as proven by the evaluations of walking track, electrophysiology, toluidine blue nerve staining, transmission electron microscopy, neural factors staining and qPCR, and gastrocnemius histology. SIGNIFICANCE: In conclusion, nerve conduit fabricated from aligned electrospinning of SPI-modified PLLA nanofibers is promising for peripheral nerve regeneration.

Structure, physical properties, hemocompatibility and cytocompatibility of starch/zein composites.

A series of composite films were prepared from glycerol-plasticized starch and zein by intensive mixing and hot press. The structure and physical properties of the starch/zein (SZ) composite films were characterized by scanning electron microscope (SEM), optical microscopy and water contract angle testing. The hemocompatibility and cytocompatibility of SZ films were evaluated by plasma recalcification time, hemolysis assay and cell culture experiment. SEM and optical observation showed that starch and zein domains can be differed in the films and in a two phase separation status. Glycerol affects the surface hydrophilicity/hydrophobicity of the films. The hemocompatibility and cytocompatibility evaluation showed that SZ composites are anticoagulant materials with no hemolysis and low cytotoxicity. The SZ composites maybe have potentials for applications as biomaterials.