Expression of basal lamina components by Schwann cells cultured on poly(lactic acid) (PLLA) and poly(caprolactone) (PCL) membranes

The present in vitro study investigated the expression of basal lamina components by Schwann cells (SCs) cultivated on PCL and PLLA membranes prepared by solvent evaporation. Cultures of SCs were obtained from sciatic nerves from neonatal Sprague Dawley rats and seeded on 24 well culture plates containing the polymer membranes. The purity of the cultures was evaluated with a Schwann cell marker antibody (anti-S-100). After one week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, laminin I and II. Positive labeling against the studied molecules was observed, indicating that such biomaterials positively stimulate Schwann cell adhesion and proliferation. Overall, the present results provide evidence that membrane-derived biodegradable polymers, particularly those derived from PLLA, are able to provide adequate substrate and stimulate SCs to produce ECM molecules, what may have in turn positive effects in vivo, influencing the peripheral nerve regeneration process.     

Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion

Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential, fibroin proteins from Antheraea mylitta and Bombyx mori are immunologically inert and invoke minimal immune response. Stimulation of murine peritoneal macrophages and RAW 264.7 murine macrophages by these fibroin proteins both in solution and in the form of films assayed in terms of nitric oxide and TNFalpha production showed comparable stimulation as in collagen. Kinetics of adhesion of L929 murine fibroblasts, for biocompatibility evaluation, monitored every 4 h from seeding and studied over a period of 24 h, reveal A. mylitta fibroin film to be a better substrate in terms of rapid and easier cellularization. Cell viability studies by MTT assay and flow cytometric analyses indicate the ability of fibroin matrices to support cell growth and proliferation comparable to collagen for long-term culture. This matrix may have potential to serve in those injuries where rapid cellularization is essential.     

The synthesis and characterization of a novel biodegradable and electroactive polyphosphazene for nerve regeneration

Conductive polymers have been of great interest to the biopharmaceutical industry because of their cell adhesion and proliferation. In this paper, a novel electrically-conductive and biodegradable polyphosphazene polymer containing parent aniline pentamer (PAP) and glycine ethyl ester (GEE) as side chains was synthesized through a nucleophilic substitution reaction for its potential application in nerve regeneration. The electrical conductivity of the polymer was ~ 2 × 10^? 5 S/cm in the semiconducting region upon preliminarily protonic-doped experiment. Degradation studies carried out in phosphate-buffered saline at 37 °C showed a mass loss of ~ 50% after 70 days. In vitro cytotoxicity to the RSC96 Schwann cells was evaluated using the cell viability assay. The polymer exhibited no cytotoxicity, indicating that such a polyphosphazene polymer has potential as scaffold material in tissue engineering for peripheral nerve regeneration or other biomedical devices that require electroactivity.     

Fiber scaffolds of polysialic acid via electrospinning for peripheral nerve regeneration

Fiber scaffolds of bioactive polysialic acid have been prepared via electrospinning for peripheral nerve regeneration. The diameter, morphology and alignment of fibers in scaffolds were adjusted by variation of electrospinning parameters, which are decisive for the cell-scaffold interaction. Due to the high water solubility of polysialic acid (poly-α-2,8-N-acetylneuraminic acid) a photoactive derivative (poly-α-2,8-N-pentenoylneuraminic acid) was used to obtain stable fiber scaffolds in water by photochemical crosslinking. At the optimized fiber scaffolds good cell viability and directed cell proliferation along the fibers was achieved by cell tests with immortalized Schwann cells.     

Chitosan/silk fibroin-based tissue-engineered graft seeded with adipose-derived stem cells enhances nerve regeneration in a rat model

Sciatic nerve injury presents an ongoing challenge in reconstructive surgery. Local stem cell application has recently been suggested as a possible novel therapy. In the present study we evaluated the potential of a chitosan/silk fibroin scaffold serving as a delivery vehicle for adipose-derived stem cells and as a structural framework for the injured nerve regeneration. The cell-loaded scaffolds were used to regenerate rat sciatic nerve across a 10 mm surgically-induced sciatic nerve injury. The functional nerve recovery was assessed by both walking track and histology analysis. Results showed that the reconstruction of the injured sciatic nerve had been significantly enhanced with restoration of nerve continuity and function recovery in the cell-loaded scaffold groups, and their target skeletal muscle had been extensively reinnervated. This study raises a potential possibility of using the newly developed nerve grafts as a promising alternative for nerve regeneration.     

Review Peripheral nerve regeneration using non-tubular alginate gel crosslinked with covalent bonds

We have developed a nerve regeneration material consisting of alginate gel crosslinked with covalent bonds. in the first part of this study, we attempted to analyze nerve regeneration through alginate gel in the early stages within 2 weeks. in the second part, we tried to regenerate cat peripheral nerve by using alginate tubular or non-tubular nerve regeneration devices, and compared their efficacies. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells, forming small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8–14 days after surgery. Remarkable restorations of the 50-mm gap in cat sciatic nerve were obtained after a long term by using tubular or non-tubular nerve regeneration material consisting mainly of alginate gel. However, there was no significant difference between both groups at electrophysiological and morphological evaluation. Although, nowadays, nerve regeneration materials being marketed mostly have a tubular structure, our results suggest that the tubular structure is not indispensable for peripheral nerve regeneration.     

Conductive Au nanowires regulated by silk fibroin nanofibers

Conductive Au-biopolymer composites have promising applications in tissue engineering such as nerve tissue regeneration. In this study, silk fibroin nanofibers were formed in aqueous solution by regulating silk self-assembly process and then used as template for Au nanowire fabrication. We performed the synthesis of Au seeds by repeating the seeding cycles for several times in order to increase the density of Au seeds on the nanofibers. After electroless plating, densely decorated Au seeds grew into irregularly shaped particles following silk nanofiber to fill the gaps between particles and finally form uniform continuous nanowires. The conductive property of the Au-silk fibroin nanowires was studied with current-voltage （I-V） measurement. A typical ohmic behavior was observed, which highlighted their potential applications in nerve tissue regeneration.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

丝素蛋白柔性电子器件材料的研究进展

丝素蛋白材料凭借良好的生物相容性、可控生物降解性、再生形貌多样性等已被制成柔性电子器件在电子领域进行了应用研究.本文首先综述不同溶解方法对蚕丝再生材料制备的影响,同时对丝素蛋白材料的(微球、膜、纤维、凝胶、支架等)制备方法、材料性能进行分析,最后总结了近年来丝素蛋白基柔性电子材料的应用研究进展.尽管已有研究表明可获得各...     

Silk RGD融合蛋白修饰羟基磷灰石/丝素蛋白支架对成骨细胞生长的影响

通过浸渍吸附的方法, 用桑蚕丝素-RGD融合蛋白(简称Silk-RGD)对多孔状磷灰石/丝素蛋白(HA/SF)复合支架材料进行表面修饰, 研究了复合支架材料在不同浓度Silk-RGD蛋白溶液中浸渍后对两种不同成骨细胞MG-63和MC3T3-E1黏附、增殖和分化的影响。结果表明, Silk-RGD融合蛋白修饰的复合支架材料的细胞黏附性能显著高于未经修饰的对照组, 且其促黏附性能具有Silk-RGD浓度依赖性; 体外培养7天时, 细胞增殖能力较对照组更显著,当Silk-RGD的吸附量为11 μg/mg时, MG-63的增殖率较对照样提高了21%, MC3T3-E1提高了50%; 而碱性磷酸酶活性检测结果显示, 复合支架经Silk-RGD表面修饰后对MC3T3-E1细胞的分化有一定的促进作用, 但对MG-63细胞的影响不明显。      

Cell Surface Engineering with Edible Protein Nanoshells

Natural protein (silk fibroin) nanoshells are assembled on the surface of Saccharomyces cerevisiae yeast cells without compromising their viability. The nanoshells facilitate initial protection of the cells and allow them to function in encapsulated state for some time period, afterwards being completely biodegraded and consumed by the cells. In contrast to a traditional methanol treatment, the gentle ionic treatment suggested here stabilizes the shell silk fibroin structure but does not compromise the viability of the cells, as indicated by the fast response of the encapsulated cells, with an immediate activation by the inducer molecules. Extremely high viability rates (up to 97%) and preserved activity of encapsulated cells are facilitated by cytocompatibility of the natural proteins and the formation of highly porous shells in contrast to traditional polyelectrolyte‐based materials. Moreover, in a high contrast to traditional synthetic shells, the silk proteins are biodegradable and can be consumed by cells at a later stage of growth, thus releasing the cells from their temporary protective capsules. These on‐demand encapsulated cells can be considered a valuable platform for biocompatible and biodegradable cell encapsulation, controlled cell protection in a synthetic environment, transfer to a device environment, and cell implantation followed by biodegradation and consumption of protective protein shells.     

Impact of Scaffold Micro and Macro Architecture on Schwann Cell Proliferation under Dynamic Conditions in a Rotating Wall Vessel Bioreactor

Over the last decade tissue engineering has emerged as a powerful alternative to regenerate lost tissues owing to trauma or tumor. Evidence shows that Schwann cell containing scaffolds have improved performance in vivo as compared to scaffolds that depend on cellularization post implantation. However, owing to limited supply of cells from the patients themselves, several approaches have been taken to enhance cell proliferation rates to produce complete and uniform cellularization of scaffolds. The most common approach is the application of a bioreactor to enhance cell proliferation rate and therefore reduce the time needed to obtain sufficiently significant number of glial cells, prior to implantation.In this study, we show the application of a rotating wall bioreactor system for studying Schwann cell proliferation on nanofibrous spiral shaped scaffolds, prepared by solvent casting and salt leaching techniques. The scaffolds were fabricated from polycaprolactone (PCL), which has ideal mechanical properties and upon degradation does not produce acidic byproducts. The spiral scaffolds were coated with aligned or random nanofibers, produced by electrospinning, to provide a substrate that mimics the native extracellular matrix and the essential contact guidance cues.At the 4 day time point, an enhanced rate of cell proliferation was observed on the open structured nanofibrous spiral scaffolds in a rotating wall bioreactor, as compared to static culture conditions. However, the cell proliferation rate on the other contemporary scaffolds architectures such as the tubular and cylindrical scaffolds show reduced cell proliferation in the bioreactor as compared to static conditions, at the same time point. Moreover, the rotating wall bioreactor does not alter the orientation or the phenotype of the Schwann cells on the aligned nanofiber containing scaffolds, wherein, the cells remain aligned along the length of the scaffolds. Therefore, these open structured spiral scaffolds pre-cultured with Schwann cells, in bioreactors could potentially shorten the time needed for grafts for peripheral nerve regeneration.     

Preparation and characterisation of zein/polyphenol nanofibres for nerve tissue regeneration

Bridging strategies are required to repair peripheral nerve injuries that result in gaps >5–8 mm. Limitations such as donor‐site morbidity and size mismatches with receptor sites for autografts, together with immunological problems associated with allografts and xenografts, have created an increased interest in the field of manufactured nerve guide conduits. In this study, zein, a plant protein‐based polymer, was electrospun to prepare nanofibrous mats. An important challenge with zein mats is the rapid change from fibre to film under aqueous conditions. Tannic acid (TA), which is a polyphenol, was selected to prepare a blend of zein/TA with different weight ratios to investigate its effect on the wetting resistance of nanofibres. The electrospun mats were characterised and evaluated by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM). Also, degradation and mechanical properties of the mats were studied. Results showed that TA had a significant effect on the resistance to film formation in nanofibres. Moreover, the degradation and elongation at break of mats were increased with increase in TA concentration. For the investigation of the peripheral nerve regeneration potential, Schwann cells were selected for cytotoxicity evaluation by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide assay and cell morphology by SEM. Schwann cells had good biocompatibility with zein/TA blends (%) of 90/10 and 80/20.Inspec keywords: polymer fibres, biomedical materials, electrospinning, cellular biophysics, tissue engineering, proteins, molecular biophysics, neurophysiology, nanofibres, injuries, nanomedicine, toxicology, scanning electron microscopy, nanofabrication, polymer blends, polymer films, wetting, Fourier transform infrared spectra, elongationOther keywords: SEM, Schwann cells, nerve tissue regeneration, peripheral nerve injuries, donor‐site morbidity, size mismatches, receptor sites, immunological problems, allografts, xenografts, manufactured nerve guide conduits, plant protein‐based polymer, nanofibrous mats, zein mats, aqueous conditions, tannic acid, wetting resistance, electrospun mats, scanning electron microscopy, film formation, TA concentration, peripheral nerve regeneration potential, cell morphology, weight ratios, zein‐polyphenol nanofibres, electrospinning, zein‐TA blends, Fourier transform infrared spectroscopy, mechanical properties, elongation‐at‐break, cytotoxicity evaluation, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide assay, biocompatibility     

Synthesis,characterization r）oly [LA-co-（GIc-alt-Lys）] for and biological evaluation of nerve regeneration scaffold

A novel nerve repairing material poly [LA-co.（GIc-alt-Lys）] （PLGL） was synthesized. The viability and growth of Schwann cells （SCs） co-cultured With poly （D, L- lactic acid） （PDLLA） films （control group） and PLGL films were evaluated by MTT assay and SEM observation. Then, contact angle measurement, histological assessment and enzyme-linked immunosorbent assay （ELISA） testing on inflammatory-related cyto- kines such as IL-10 and TGF-β1 were performed. The results showed that, compared with PDLLA, PLGL films possesses better hydrophilicity, biocompatibility, degradation property and less inflammatory reaction. The present study indicated that PLGL scaffolds would meet the requirements of artificial nerve scaffold and have a potential application in the fields of nerve regeneration.     

Silk fibroin/chitosan scaffold: preparation,characterization, and culture with HepG2 cell

Tissue engineering requires the development of three-dimensional water-stable scaffolds. In this study, silk fibroin/chitosan (SFCS) scaffold was successfully prepared by freeze-drying method. The scaffold is water-stable, only swelling to a limited extent depending on its composition. Fourier Transform Infrared (FTIR) spectra and X-Ray diffraction curves confirmed the different structure of SFCS scaffolds from both chitosan and silk fibroin. The homogeneous porous structure, together with nano-scale compatibility of the two naturally derived polymers, gives rise to the controllable mechanical properties of SFCS scaffolds. By varying the composition, both the compressive modulus and compressive strength of SFCS scaffolds can be controlled. The porosity of SFCS scaffolds is above 95% when the total concentration of silk fibroin and chitosan is below 6 wt%. The pore sizes of the SFCS scaffolds range from 100 μm to 150 μm, which can be regulated by changing the total concentration. MTT assay showed that SFCS scaffolds can promote the proliferation of HepG2 cells (human hepatoma cell line) significantly. All these results make SFCS scaffold a suitable candidate for tissue engineering.     

Engineered spider silk-based 2D and 3D materials prevent microbial infestation

Biofilm formation, especially of antimicrobiotic-resistant microbial strains, are a major problem in health care. Therefore, there is great interest in developing advanced materials that are selectively inhibiting microbial adhesion to surfaces, but at the same time promoting mammalian cell growth. In nature, some spider silks have evolved to repel microbes, a feature that could be used in biomaterials. To unravel how microbe repellence can be achieved in engineered spider silk, different recombinant spider silk proteins based on the consensus sequences of Araneus diadematus dragline silk proteins (fibroin 3 and 4) were processed into 2D-patterned films and 3D-hydrogels. Strikingly, protein structure characteristics on the nanoscale are the basis for the detected microbe-repellence. Designed spider silk materials promoted mammalian cell attachment and proliferation while inhibiting microbial infestation, demonstrating the great potential of these engineered spider silk-based materials as bio-selective microbial-resistant coatings in biomedical as well as technical applications.     

A quicker degradation rate is yielded by a novel kind of transgenic silk fibroin consisting of shortened silk fibroin heavy chains fused with matrix metalloproteinase cleavage sites

Degradation performance of silk fibroin is an important property for its medical applications. Herein we constructed a shortened silk fibroin heavy chain protein fused with a matrix metalloproteinase cleavage site (SSFH-MMP) along with a glutathione S-transferase tag ahead. The digestion assay shows it can be cut by matrix metalloproteinase-2 (MMP-2) at its MMP cleavage site. Furthermore, we introduced the SSFH-MMP into silk fibroin by genetic modification of silkworms in order to increase the degradation rate of the silk fibroin. After acquisition of a race of transgenic silkworms with the coding sequence of the MMP cleavage site in their genomic DNA, we tested some properties of their silk fibroin designated TSF-MMP. The results show that the TSF-MMP has MMP cleavage sites and yields a quicker degradation rate during dilution in MMP-2 enzyme buffer or implantation into tumor tissues compared with that of normal silk fibroin. Moreover, the TSF-MMP is in vitro non-toxic to human bone marrow mesenchymal stem cells (hBM-MSCs) indicating that the TSF-MMP may become a biomaterial with a quicker degradation rate for its medical applications.     

Characterization of PC12 cell proliferation and differentiation-stimulated by ECM adhesion proteins and neurotrophic factors

Among the various elements which influence axonal outgrowth in vivo is the physicochemical interaction of actively outgrowing nerve fibers with the various substrata they encounter during differentiation. Several experiments have explored the role of the extracellular matrix (ECM) in the control of neuronal differentiation. The nature, however, of the interactions between neurons and components of the ECM during regeneration and development are largely a matter of speculation. Although previous studies have already explored the influence of a number of ECM adhesion proteins and neurotrophic factors on neurite outgrowth, none have been carried in a systematic approach that allows for the simultaneous comparison of different surface conditions in relation to different neurotrophic factors. Motivated by the necessity of establishing controlled environments that allow for the rational design of stable neuronal/biomaterial interfaces, the long-term effects of NGF and FGF-2 on the behavior of PC12 cells plated on collagen and laminin modified surfaces were evaluated. A pheochromocytoma cell line derived from transplantable rat adrenal medulla, PC12 cells have been commonly employed as an instructive model for studying the underlying mechanisms of neuronal differentiation. Long-term characterization of PC12 proliferation and neuronal differentiation for an experimental duration of 7–22 days was achieved by both qualitatively and quantitatively assaying for cell count, neurite number, neurite mean length, and neurite stability. Neurite stability was determined in terms of resistance to loss after neurotrophic factor (NGF/FGF-2) withdrawal. The present findings demonstrate that ECM adhesion proteins collagen and laminin are equally effective in promoting PC12 proliferation. It was noted as well that NGF supplemented collagen cultures are significantly more efficient in providing long-term support to PC12 differentiation in terms of neurite number, mean length, and stability.     

Bovine pericardium coated with biopolymeric films as an alternative to prevent calcification: In vitro calcification and cytotoxicity results

Bovine pericardium, for cardiac valve fabrication, was coated with either chitosan or silk fibroin film. In vitro calcification tests of coated and non coated bovine pericardium were performed in simulated body fluid solution in order to investigate potential alternatives to minimize calcification on implanted heart valves. Complementary, morphology was assessed by scanning electron microscopy — SEM; X-ray diffraction (XRD) and infrared spectroscopy (FTIR-ATR) were performed for structural characterization of coatings and biocompatibility of chitosan. Silk fibroin films were assayed by in vitro cytotoxicity and endothelial cell growth tests. Bovine pericardium coated with silk fibroin or chitosan did not present calcification during in vitro calcification tests, indicating that these biopolymeric coatings do not induce bovine pericardium calcification. Chitosan and silk fibroin films were characterized as non cytotoxic and silk fibroin films presented high affinity to endothelial cells. The results indicate that bovine pericardium coated with silk fibroin is a potential candidate for cardiac valve fabrication, since the affinity of silk fibroin to endothelial cells can be explored to induce the tissue endothelization and therefore, increase valve durability by increasing their mechanical resistance and protecting them against calcification.     

Fabrication and characterization of <Emphasis Type="Italic">Antheraea pernyi</Emphasis> silk fibroin-blended P(LLA-CL) nanofibrous scaffolds for peripheral nerve tissue engineering

Electrospun nanofibers have gained widespreading interest for tissue engineering application. In the present study, ApF/P(LLA-CL) nanofibrous scaffolds were fabricated via electrospinning. The feasibility of the material as tissue engineering nerve scaffold was investigated in vitro. The average diameter increased with decreasing the blend ratio of ApF to P(LLA-CL). Characterization of ¹³C NMR and FTIR clarified that there is no obvious chemical bond reaction between ApF and P(LLA-CL). The tensile strength and elongation at break increased with the content increase of P(LLA-CL). The surface hydrophilic property of nanofibrous scaffolds enhanced with the increased content of ApF. Cell viability studies with Schwann cells demonstrated that ApF/P(LLA-CL) blended nanofibrous scaffolds significantly promoted cell growth as compare to P(LLA-CL), especially when the weight ratio of ApF to P(LLA-CL) was 25:75. The present work provides a basis for further studies of this novel nanofibrous material (ApF/P(LLA-CL)) in peripheral nerve tissue repair or regeneration.