www.nature.com/scientificreports OPEN Lycium barbarum polysaccharide encapsulated Poly lactic-co-glycolic acid Nanofibers: cost effective Received: 27 September 2017 herbal medicine for potential Accepted: 14 May 2018 Published: xx xx xxxx application in peripheral nerve tissue engineering 1,2 2 3,5 2 2,4 4,5 Jing Wang , Lingling Tian , Liumin He , Nuan Chen , Seeram Ramakrishna , Kwok-Fai So & Xiumei Mo Nerve regeneration is a serious clinical challenge following peripheral nerve injury. Lycium barbarum polysaccharide (LBP) is the major component of wolfberry extract, which has been shown to be neuroprotective and promising in nerve recovery in many studies. Electrospun nanofibers, especially core-shell structured nanofibers being capable of serving as both drug delivery system and tissue engineering scaffolds, are well known to be suitable scaffolds for regeneration of peripheral nerve applications. In this study, LBP was incorporated into core-shell structured nanofibrous scaffolds via coaxial electrospinning. Alamar blue assays were performed to investigate the proliferation of both PC12 and Schwann cells cultured on the scaffolds. The neuronal differentiation of PC12 cells was evaluated by NF200 expression with immunostaining and morphology changes observed by SEM. The results indicated that the released LBP dramatically enhanced both proliferation and neuronal differentiation of PC12 cells induced by NGF. Additionally, the promotion of Schwann cells myelination and neurite outgrowth of DRG neurons were also observed on LBP loaded scaffolds by LSCM with immunostaining. In summary, LBP, as a drug with neuroprotection, encapsulated into electrospun nanofibers could be a potential candidate as tissue engineered scaffold for peripheral nerve regeneration. Peripheral nerve injury (PNI) caused by accidents, physical conflict and surgical intervention is a common global clinical problem which can significantly ae ff ct the patients’ quality of life. Over 200 thousand procedures were performed in the US alone annually to repair PNI, and it cost 150 billion dollars annually in the US, which cause an enormous socioeconomic burden . Although the peripheral nervous system (PNS) has a capacity for axonal regeneration aer ft injury, axonal reconnection and functional recovery by spontaneous peripheral nerve repair are nearly always incomplete. In recent hundred years, various types of medical therapy have been used to repair nerve lesions. Normally, the surgical reconnection is possible for bridging small PNI gaps where the disconnected nerve stumps can be State Key Laboratory of Modification of Chemical Fibers and Polymer Materials, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China. Center for Nanofibers and Nanotechnology, E3-05-14, Department of Mechanical Engineering, Faculty of Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore, 117576, Singapore. Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China. Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, 510632, China. Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, 510632, China. Correspondence and requests for materials should be addressed to K.-F.S. (email: firstname.lastname@example.org) or X.M. (email: email@example.com) Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 1 www.nature.com/scientificreports/ 2 3 sutured end-to-end . However, when the defect is larger (>10 mm in rats, or >30 mm in humans) , retracts after injury and tensionless repair is impossible . In this case, graft between nerve stumps is required to bridge the gap and support axonal regrowth. The implantation of autologous nerve graft is regard as the gold standard therapy for peripheral nerve gap repair . However, several drawbacks of this therapy such as the need for multiple surgeries, and donor site morbidity limit its widespread clinical use, especially for longer nerve gaps. With the progress in tissue engineering, recent advances in neural tissue engineering have shown great promise for neural regeneration. Various biological and artificial nerve grafts have been used as supplement and even substitution for autologous nerve grafts. A typical tissue engineered nerve graft is composed a biomaterial-based scao ff ld and a multitude of cellular and/or molecular components . Generally, the scao ff lds are capable of guiding the regeneration of axons and function as a bridge to restore the gap. Additionally, the biomaterial-based scao ff ld should mimic the native extracellular matrix (ECM) structure, which can provide enough space and adhesion sites for the growth of cells and extension of axons. What’s more, the scao ff ld should have the capacity to deliver drugs and/or signaling factors which can direct the growth and extension of regenerating axons. Electrospun nanob fi ers have been used extensively as potential scao ff ld in neural tissue engineering . It pro- vides a conducive environment for cellular functions including adhesion, migration, proliferation and differ - entiation due to its close imitation of native neural ECM. On the other hand, core-shell structured nanofibers 6,7 fabricated via coaxial electrospinning has frequently been served as a carrier for drug release application . Proteins, drugs, and nucleic acids all have been delivered by core-shell nanob fi ers. The drug release behavior can be controlled by turning the core-sheath compositions, while retaining their bioactivity. Meanwhile, nanob fi ers exhibit much higher surface area-to- volume ratio which will facilitate the release the release of drugs and increase 8–10 the contact area between cells and fibers, thereby enhancing drug uptake by cells . Hence, electrospun nano- b fi ers are potential candidates for nerve regeneration applications. Poly (lactic-co-glycolic acid) (PLGA) has been 11–13 used in many tissue engineering application due to its biodegradability and biocompatibility . Electrospun PLGA nanob fi ers have also been used to enhance the sciatic nerve regeneration in rat models . Bioactive molecule is another important factor in tissue engineering, which can improve neurogenesis in nerve injury site. However, most bioactive molecules such as growth factors and cytokines are expensive and instable in clinic efficacy, sometimes have side effects . Plant extracts provide an alternative choice which always have multi-target and less side effects. And the plant extracts used for nerve repair should be effective in axonal regeneration and functional recovery. Wolfberry extracts is made up of a group of components including poly- saccharides, betaine, zeaxanthin, beta-carotene, groups of vitamin, 19 kinds of amino acid and trace minerals . It is believed that the polysaccharides of wolfberry, also named as Lycium barbarum polysaccharides (LBP), are the active components responsible for its various pharmacological properties including anti-aging, anti-tumor, 17,18 anti-oxidation and immune modulation effects . Increasing lines of evidence discloses the therapeutic effects of LBP on neuroprotection, which raises the possibility of using LBP as a neuroprotective agent aer n ft erve inju- ries . Studies involving primary cultured cortical neurons showed that LBP treatment reduced cellular death 19–21 induced by amyloid-beta peptides and homocysteine insults . Chan et al. reported that intragastric admin- istration of LBP can reduce the death of retinal ganglion cells induced by experimental ocular hypertension . Lau et al. found that LBP could promote the neuronal proliferation, differentiation of neural precursor cells . Studies of Gao et al. also found that LBP could effectively protect PC12 cells and primary neurons against 6-hydroxydopamine-induced cell death . Thus, introduction of bioactive chemicals such as LBP in neural tissue engineering may accelerate nerve regeneration. In our present study, we intended to investigate the effects of LBP along with PLGA nanofibrous scao ff ld on nerve cells behaviors such as proliferation, neuronal die ff rentiation, neurite outgrowth and myelination in vitro. For the first time, we evaluated the effect of LBP on PC12 cells and Schwann cells proliferation and the differentiation of PC12 cells. In order to further confirm the potential role of LBP in peripheral nerve regeneration, core-shell structured PLGA-LBP nanob fi ers loading different dosage of LBP were fabricated via coaxial electrospinning. The topography of nanofibers was characterized by Scanning electron microscope (SEM), while the core-shell structure was confirmed by Transmission electron microscope (TEM) and fluorescent images. The surface hydrophilicity were evaluated by water contact angle. The mechanical properties of the scao ff ld were measured with tensile mechanical test. The in vitro release behavior of LBP from core-shell structured PLGA-LBP nanob fi ers was also determined. Then biocompatibility of the scao ff ld and the influence of scaffolds with diverse amount of LBP on nerve cells growth were tested in terms of proliferation and morphology of PC12 cells and Schwann cells. Furthermore, the neuronal differentiation of PC12 cells were performed on the scaffold in the absence and presence of Nerve growth factor (NGF). In order to explore the potential application of the PLGA-LBP nanob fi rous scao ff ld in peripheral nerve repair, the neurite outgrowth of Dorsal root ganglion (DRG) neurons and the myelination of Schwann cell were further investigated. Results The objective of this research was to develop a coaxial electrospun nanofibrous scao ff ld containing LBP, which might improve both the differentiation of neurons and myelination of Schwann cells for peripheral nerve regen- eration. The preparation process and working hypothesis of the scao ff ld are schematically illustrated in Fig. 1. Fabrication of PLGA-LBP nanofibrous scaffolds. Coaxial electrospinning was utilized to fabricate PLGA-LBP nanofibrous scao ff ld during this study. PLGA-LBP10, PLGA-LBP30, and PLGA-LBP50 were fabri- cated with LBP aqueous solution of three different concentration as the core solution. PLGA-LBP0 was fabricated with pure PLGA solution by conventional electrospinning. The fabrication process is schematically demonstrated in Fig. 1a. Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 2 www.nature.com/scientificreports/ Figure 1. Schematic illustration of the preparation process and working hypothesis for coaxial electrospun b fi er scao ff lds. (a) The setup of coaxial electrospinning: the spinneret is composed of two concentric needles; the outer needle is used to deliver the shell solution (blue), while the inner needle is used to eject the core solution (Pink). (b) The LBP encapsulated in the core of coaxial fibers could be released in a sustained manner to +NGF promote differentiation of PC12 cells with Differentiation Medium . (c) Scao ff lds coated with Poly-L-lysine could promote the adhesion of Schwann cells, and the released LBP could enhance the myelination of Schwann cells. (d) The neurite outgrowth of DRG neurons might be improved under the synergistic effect of LBP and NGF. Physico-chemical properties of nanofibrous scaffolds. Surface morphology of nanobr fi ous scaffolds . In nerve tissue, the topographical features of fibers play a significant role towards the behavior of cells. In Fig. 2a, the SEM images of the resultant nanofibers show the effects of various contents of LBP on nanofiber topo- graphic features. It can be preliminary observed that all the nanofibers were randomly oriented, uniform and smooth in appearance without beads, indicating that LBP distributed uniformly in PLGA nanob fi ers. According to Fig. 2a and Table 1, the diameters of PLGA-LBP0, PLGA-LBP10, PLGA-LBP30 and PLGA-LBP50 were 896.35 ± 200.19 nm, 379.14 ± 85.39 nm, 305.88 ± 62.11 nm, and 295.90 ± 67.85 nm, indicating that incorpora- tion of LBP caused great reduction in fiber diameters. Additionally, with the increase of LBP concentration, the average diameters of fibers decreased accordingly. The main reason is that the electric conductivity of electrospun suspensions increased with the increased content of LBP. Core-shell structure of nanob fi rous scao ff lds. e Th core-shell structure of LBP encapsulated nanob fi ers was con- firmed by TEM and fluorescent images which were shown in Fig. 2b,c. The TEM image clearly indicated the formation of the core-shell structure which confirmed the incorporation of LBP within the polymeric shell. Fluorescent image showed the green light emitted from nanob fi ers duo to the existence of fluorescein isothiocy- anate (FITC), suggesting that the mixture of LBP and FTIC was loaded inside the core of nanob fi ers successfully. e Th uniformly distributed green light along these b fi ers indicated the uniform dispersion of LBP along nanob fi er axis. e Th mechanical properties of nanob fi rous scao ff lds. Peripheral nerves are specific viscoelastic tissues with high mechanical properties , so mechanical properties of nanofibers would play critical roles in peripheral nerve applications. The potential scao ff lds should provide sufficient mechanical support for cells and axon regeneration. From Table 1 and Fig. S1, the tensile stress for PLGA-LBP0, PLGA-LBP10, PLGA-LBP30 and PLGA-LBP50 were 5.06 ± 0.21, 5.03 ± 0.52, 4.22 ± 0.43, 3.93 ± 0.39 MPa. Borschel et al. reported that stress of fresh rat sciatic nerve was 2.72 ± 0.97 MPa, therefore the PLGA-LBP nanofibers had sufficient tensile stress to be utilized as a nerve graft. As shown in Fig. S1, there was no significant difference between the tensile stress of scao ff lds PLGA-LBP0 and PLGA-LBP10. However, the values of scao ff lds PLGA-LBP30 and PLGA-LBP50 decreased gradually with the increasing content of LBP. As for the breaking elongations for different scaffolds, the values represented a Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 3 www.nature.com/scientificreports/ Figure 2. (a) SEM images of nanob fi ers and their diameter distributions. The scale bar is 10 μm; (b) TEM image of PLGA-LBP30 nanob fi ers; (c) Fluorescent image of PLGA-LBP30 nanob fi ers with FITC. Mechanical properties Water contact Breaking Diameter angle tensile stress Breaking Young’s Scaffold (nm/n = 100) (°/n = 3) (MPa) elongation (%) Modulus (MPa) PLGA-LBP0 896.35 ± 200.19 135.4 ± 4.4 5.06 ± 0.21 236.92 ± 69.70 200.79 ± 16.02 PLGA-LBP10 379.14 ± 85.39 135.8 ± 2.8 5.03 ± 0.52 26.73 ± 4.81 163.84 ± 11.08 PLGA-LBP30 305.88 ± 62.11 135.3 ± 4.1 4.22 ± 0.43 24.18 ± 5.92 153.03 ± 11.58 PLGA-LBP50 295.90 ± 67.85 135.8 ± 1.9 3.93 ± 0.39 18.97 ± 6.45 138.95 ± 10.18 Table 1. Diameter, water contact angle and mechanical properties of scao ff lds. great reduction from 236.92 ± 69.70% for PLGA-LBP0 to 18.97 ± 6.45% for PLGA-LBP50. The Young’s mod- ulus decreased from 200.79 ± 16.02 MPa for PLGA-LBP0 to 138.95 ± 10.18 MPa for PLGA-LBP50, and there was a significant die ff rence between scao ff lds containing LBP and pure PLGA scao ff lds. Compared to scao ff lds PLGA-LBP10 and PLGA-LBP30, the decrease of Young’s modulus for PLGA-LBP50 was also statistically signifi- cant. The mechanical properties of nanob fi rous scao ff ld decreased with the increased content of LBP, which was consistent with published results - the addition of low molecular drug had a “plasticizing” effect to fibers . Even the mechanical properties decreased with the addition of LBP, the PLGA-LBP nanofibrous scaffolds were still suitable for nerve tissue engineering applications. e s Th urface hydrophilicity of nanob fi rous scao ff ld. Water contact angle of nanob fi rous scao ff ld reflects its surface hydrophilicity, which were shown in Table 1 and Fig. 3a. The water contact angles of PLGA-LBP0, PLGA-LBP10, PLGA-LBP30 and PLGA-LBP50 scao ff lds were 135.4° ± 4.4°, 135.8 ± 2.8°, 135.3° ± 4.1° and 135.8° ± 1.9°. PLGA is a hydrophobic polymer, and the nanob fi rous scao ff ld based on PLGA was also hydrophobic. LBP encapsulated inside the polymer matrix wouldn’t change the nanob fi ers surface hydrophilicity, so there was no obvious differ - ence among these four different scao ff lds in surface hydrophilicity. In vitro release behavior of LBP from core-shell nanofibers. The in vitro release behavior of drugs is ae ff cted by several factors including the degradation rate of the carriers, hydrophilicity of drugs and the drug dis- tribution. In our study, coaxial electrospinning was used to fabricate core-shell structured nanob fi ers, which could Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 4 www.nature.com/scientificreports/ Figure 3. (a) Water contact angles of four different nanob fi rous scao ff lds. A: PLGA-LBP0 B: PLGA-LBP10 C: PLGA-LBP30 D: PLGA-LBP50. The data represent the mean of three independent experiments (n = 3, mean ± SD) (b) In vitro LBP release profile from PLGA-LBP30 nanob fi rous scao ff lds. The data represent the mean of three samples (n = 3, mean ± SD) (c) Typical SEM images of PLGA-LBP0, PLGA-LBP10, PLGA-LBP30 and PLGA-LBP50 nanob fi rous scao ff lds aer deg ft radation in PBS at 37 °C. The scale bar is 20 μm. encapsulate drugs in the core. e Th shell part could alleviate burst release and preserve the bioactivity of drugs as a barrier. Stimulating nerve growth at the injury site is most important during the first 10–15 days of repair , and approximately 4 weeks were also very crucial for the peripheral nerve regenerations, which has been verified in rat sciatic nerve . Hence, 4 weeks release of the LBP incorporated in the fibers would be favorable. The Ultra-violet absorption spectra of LBP solution with various concentration and the calculated standard line of LBP were shown in Figs S2 and S3, respectively. According to the accumulation release curve shown in Fig. 3b, the LBP exhibited a sustained release lasted for at least 2 months. The release kinetics can be described in two stages: a fast burst release (the first 7 days) followed by a sustained and constant release (the following 53 days). The initial fast release resulted from diffusion of LBP located near the surface of the fibers. The fast release for the first day was about 10%, which followed by a release until 7 days with a cumulative release of 40%. Ae ft r 7 days, LBP displayed a sustained release over several weeks due to the increased diffusion path with the slow degradation of PLGA. During the period of 60 days of release, the cumulative release of LBP reached over 70%. In summary, the release results of LBP from PLGA-LBP30 nanob fi rous scao ff lds could satisfy the requirement in peripheral nerve regeneration. The in vitro degradation of nanofibrous scaffolds. e m Th orphologies of PLGA, PLGA-LBP10, PLGA-LBP30 and PLGA-LBP50 nanofibrous scaffolds after 1, 3, 6, 8 weeks degradation were shown in Fig. 3c. All nanofibers remained the fibrous structures aer 3 w ft eeks degradation. Aer 6 w ft eeks degradation, PLGA-LBP10, PLGA-LBP30 and PLGA-LBP50 started losing the fibrous structures, while the PLGA nanob fi ers still kept apparent fibrous structure with swollen appearance. Aer 8 w ft eeks degradation, the fibrous structures of all scao ff lds were blurred. The results indicated that the incorporation of LBP ae ff cted the degradation rate of scao ff lds. With the release of LBP, more phosphate buffer solution (PBS) could enter inside the fiber and accelerated the degradation of scao ff lds. Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 5 www.nature.com/scientificreports/ Figure 4. Proliferation of (a) PC12 cells and (b) Schwann cells on different scao ff lds. The data represent the mean of three samples (n = 3, mean ± SD). *Denotes significance against all the other groups using one-way ANOVA test; denotes significance between two groups using one-way ANOVA test, P < 0.05. The cell behavior of PC12 cells on scaffolds. e p Th roliferation of PC12 cells. In various studies, PC12 cells have been used as neuron precursors . Therefore, the proliferation of PC12 cells should be considered in peripheral nerve tissue engineering. The ability of a tissue engineered scao ff ld to support the proliferation of cells is one of the major criteria to determine its application in target tissue regeneration. As shown in Fig. 4a, PC12 cells could attach, spread, and proliferate on all the nanob fi rous scao ff lds 1 day ae ft r seeding. However, the cell proliferation showed difference on different scao ff lds on day 3, day 5 and day 7. PLGA-LBP30 nanob fi rous scao ff lds were more favorable for cell proliferation, which indicated that there was an optimal dosage for LBP used in peripheral nerve regenera- tion. Above all, the proliferation of PC12 was promoted by released LBP from nanob fi rous scao ff lds. Neuronal dier ff entiation of PC12 cells. PC12 cells are the most widely used cells for the neurogenesis study. The morphology changes of PC12 cells will reflect its differentiation directly. Figure 5a displayed the morphology of undifferentiated PC12 cells, which was smaller and spherical on all nanob fi rous scao ff lds. Aer ft being treated with Differentiation Medium with/without NGF for 10 days, the morphology of PC12 cell changed (Fig. 5b). A few cells –NGF grown on PLGA-LBP30 and PLGA-LBP50 nanob fi rous scao ff lds with Differentiation Medium showed elongated +NGF shape, while on PLGA-LBP10 and PLGA-LBP30 nanob fi rous scao ff lds with Differentiation Medium , there were more cells showed a phenotype with elongated morphologies. Additionally, some cells on PLGA-LBP0 nanob fi rous +NGF −NGF scao ff lds with Differentiation Medium also projected the neurites, while with Differentiation Medium , the cells were still showed undifferentiated phenotype. The phenotype of elongated morphologies confirmed the differenti- ation of PC12 cells. From all the aforementioned results, we concluded that NGF was essential in stimulating PC12 cells differentiation, but the stimulation of NGF could be enhanced with moderated amount of LBP. Neurofilaments (NF) are the major groups of intermediated filaments which are found predominantly in cells and tissues of neuronal origin. Neurofilament proteins are synthesized in the neuronal perikarya, assembled to form filaments and then slowly transported within the axons towards the synaptic terminals . Based on the results of PC12 cells proliferation and morphology of die ff rentiated PC12 cells, we found that PLGA-LBP30 nano - b fi rous scao ff lds should be more effective on nerve regeneration. Therefore, the expression of NF200 was evalu- ated to further confirm the neuronal differentiation of PC12 cells on PLGA-LBP30, and PLGA-LBP0 was used as control. The immunostaining of NF200 also enabled the comparison of cell phenotype and neurite outgrowth on different nanofibrous scao ff lds. It can be seen from Fig. 5c that the results were consistent with morphology observation by SEM. The cells cultured on both scaffolds with Differentiation Medium with/without NGF all expressed NF200 protein, but the PLGA-LBP30 scao ff lds showed more supportive for NF200 expression com- pared to cell differentiation observed on PLGA-LBP0 in Differentiation Medium with NGF. Most PC12 cells on PLGA-LBP30 nanob fi rous scao ff lds showed a neuro-like appearance with bigger soma and well grown neurites in Die ff rentiation Medium with NGF. Meanwhile, PC12 cells on PLGA-LBP0 scao ff lds in Differentiation Medium with NGF showed more neurites formed compared to that in Differentiation Medium without NGF. These results also indicated that NGF was an essential stimulation for PC12 differentiation, and the synergistic effect of LBP and NGF on neuronal differentiation of PC12 cells could be more effective. Cell behavior of Schwann cells and DRG neurons on scaffolds. The proliferation of Schwann cells. Schwann cells play a critical role in neuron survival and regeneration. They can provide neurotropic growth factors such as NGF and neurotrophin 3 (NT3) for neurite outgrowth as well as axonal nerve regenera- 32,33 tion . Additionally, the proliferation of Schwann cells can form columns of cells, known as brands of Büngner, which serve as a physical guide for regenerating nerve axons . Therefore, the proliferation of Schwann cells should also be considered in peripheral nerve tissue engineering. According to Fig. 4b, the proliferation of Schwann cells showed difference aer 1 d ft ay seeding. For all four time points, LBP contained nanob fi rous scao ff lds resulted in better cell proliferation, especially the PLGA-LBP10 nanob fi rous scao ff lds. The results suggested that the optimal dosage of LBP for Schwann cell proliferation was slightly lower than that for PC12 cells. Above all, the proliferation of Schwann cells was also promoted by released LBP from nanob fi rous scao ff lds. Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 6 www.nature.com/scientificreports/ Figure 5. (a) Morphology of PC12 cells cultured with growth medium before differentiation; (b) Morphology of PC12 cells aer diff ft erentiation on different nanob fi rous scao ff lds with different Differentiation Medium; (c) NF200 expression of PC12 cells on different nanob fi rous scao ff lds with different Differentiation Medium. The red scale bar is 20 μm. (+NGF: The cells cultured with Differentiation Medium with NGF; −NGF: The cells cultured with Differentiation Medium without NGF. The red arrow indicated the cell elongation). e m Th orphology study of Schwann cells. It has been known that Schwann cells play a critical role via the syner- 35–37 getic effect with neurons and macrophages during the regeneration process of injured PNS . Thus to further confirm the interactions of Schwann cells with electrospun nanob fi rous scao ff lds, the morphology and phenotype of cells were evaluated aer ft 7 days in vitro culture by SEM and immunostaining. e Th SEM images in Fig. 6 demon- strate the interaction of Schwann cells with four different nanofibrous scaffolds. The cells interacted and inte- grated well with all scao ff lds, and Schwann cells exhibited a typical spindle-like shape with bio-polar extensions. Notably on LBP loaded nanob fi rous scao ff lds, Schwann cells grew in higher density, and the interaction between cell-cell and cell-scao ff ld was much better than pure PLGA scao ff lds, especially on PLGA-LBP30 scao ff lds. In all, the growth of Schwann cells was enhanced with LBP released from the scao ff lds, and the PLGA-LBP30 scao ff lds was the best choice for application in nerve regeneration. In order to corroborate the results obtained by SEM observation, immunostaining with S-100 antibody, which was a specific protein for Schwann cells was conducted. According to the aforementioned results, PLGA-LBP30 and PLGA-LBP0 scao ff lds were used for this evaluation, and tissue culture plate (TCP) was chosen as control. Confocal images presented in Fig. 6 confirmed that the cells showed a higher proliferation on PLGA-LBP30 scaf- folds. Compared to cells on TCP, the Schwann cells exhibited higher maturation on both nanofibrous scao ff lds especially on PLGA-LBP30. All these results indicate that the PLGA-LBP30 scao ff lds were the most favorable for both PC12 and Schwann cells growth. Therefore PLGA-LBP30 scao ff ld was selected for further study, and PLGA-LBP0 scao ff ld was used as control. Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 7 www.nature.com/scientificreports/ Figure 6. SEM images of Schwann cells on different nanob fi rous scao ff lds and confocal micrographs of Schwann cells on (a) TCP, (b) PLGA-LBP0 and (c) PLGA-LBP30 nanob fi rous scao ff lds aer 7 d ft ays of in vitro culture. Myelin basic protein (MBP) expression of Schwann cells. Myelination of Schwann cells is critical for the suc- cess of peripheral nerve regeneration. The matured myelinating Schwann cells are able to secrete various neu- rotrophins such as NGF, brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), 38,39 which can promote the neurogenesis and guide axon elongation . To investigate the myelinating of Schwann cells, the expression of marker of Schwann cells myelination, such as myelin basic protein (MBP) was measured with immunostaining. As shown in Fig. 7, the cells on both scaffolds expressed MBP, but obviously stronger uo fl rescence was observed in cells on PLGA-LBP30 scao ff lds. Schwann cells showed a mature bipolar shape and arranged in bundles in PLGA-LBP30 scao ff lds. As we know, the Schwann cells usually migrate and form a column of cells known as brands of Bünger to guide the regeneration of axon aer n ft erve injuries. So PLGA-LBP30 nano- b fi rous scao ff lds have the potential to enhance the myelination of Schwann cells in peripheral nerve regeneration. Neurite outgrowth of DRG neurons on scaffolds. We have demonstrated that LBP loaded PLGA nanofibrous scaffolds promoted proliferation and neurite outgrowth of PC12 cells. For further study, we performed eval- uation of neurite outgrowth behavior of the rat primary DRG neurons. The expression of Neurofilament 160 protein (NF160) was evaluated by immunostaining and observed by LSCM. The results in Fig. 8a revealed that NF160 expression was significantly improved in the PLGA-LBP30 scao ff lds. We also found that the neurite out- growth of the DRG neurons on PLGA-LBP30 scao ff lds was faster than that on PLGA-LBP0 scao ff lds. To deter - mine whether the LBP encapsulation in the scao ff lds influenced the extent of neurite growth or not, the lengths of all of the representative neurites were measured using ImageJ software (Fig. 8b). The neurite length is defined as the linear distance from the tip of the neurite to the cell junction. The average neurite length of DRG neurons Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 8 www.nature.com/scientificreports/ Figure 7. MBP expression of Schwann cells. Immunouo fl rescence images of Schwann cells cultured on PLGA- LBP0 and PLGA-LBP30 scao ff lds for 10 days (Green: MBP; Blue: Nuclei stained with DAPI). on PLGA-LBP0 scao ff lds was 35.62 ± 11.83 μm, while the value increased to 120.17 ± 45.44 μm for DRG neurons cultured on PLGA-LBP30 scao ff lds. Moreover, the longest neurite was up to 251.51 μm on PLGA-LBP30 scao ff lds. Comparison of the mean neurite lengths of the DRG neurons cultured on PLGA-LBP30 scaffolds with those of neurons cultured on PLGA-LBP0 scao ff lds revealed that the axon elongation was significantly enhanced on PLGA-LBP30 scao ff lds. This may be attributed to the released LBP from the scao ff lds. Discussion Wolfberry has been used as functional food as well as medicinal herbs in Asian countries for more than thou- sands of years. LBP as the active component extracted from Wolfberry has been shown to be neuroprotective in many studies . Electrospun nanob fi ers have been widely used as scao ff ld for peripheral nerve tissue engineering application. A variety of natural and synthetic polymers have been fabricated into nanofibers to support neural tissue engineering development. PLGA, as a biocompatible and biodegradable polymer material, has been widely 15,40 used in peripheral tissue engineering . The present study was intended to fabricate PLGA nanofibers loaded with LBP via coaxial electrospinning. Thus, a synergistic effect was produced by the combination of nerve tissue engineering and drug therapy to accelerate the nerve regeneration. In the present study, four die ff rent groups of nanob fi ers were prepared according to the die ff rent concentra - tion of LBP. The diameters of fibers gradually decreased from about 900 nm to about 300 nm with the increase of LBP concentration (Fig. 2). Early studies have reported that randomly oriented nanob fi ers with smaller diameters (~300 nm) are more favorable for nerve cells growth compared to nanofibers with large diameters (~750 nm) . e LB Th P was indeed encapsulated within the polymer shell and could be released slowly from the fibrous scao ff ld (Fig. 3b). The feasibility of LBP encapsulated nanob fi ers for peripheral nerve tissue engineering application was evaluated by identification of nerve cell behaviors on die ff rent nanob fi ers. PC12 cells are derived obtained from a pheochromocytoma of the rat adrenal medulla, which are widely used as model cells in neurophysiological and neuropharmacological studies . Schwann cells are the glial cells in peripheral nerve system, which play a key role in Wallerian degeneration and subsequent regeneration, and provide a suitable environment for regeneration. In this study, the proliferation of both PC12 and Schwann cells were significantly enhanced in LBP containing nanob fi ers, especially on PLGA-LBP30 group (Fig. 4). Yu et al. found that LBP could prevent neuronal death via both necrosis and apoptosis with a wide range of effective dosages in vitro by inhibiting two key pro-apoptotic signaling pathways which were c-jun N-terminal kinase (JNK) and double-stranded-RNA-dependent protein 19,20 kinase (PKR) . Additionally, Ho et al. reported that cell death of Rat primary cultured cortical neurons treated with Aβ and Aβ was reduced by LBP through the improve of phosphorylation of Akt . Consequently, 25–35 1–42 the application of LBP loaded nanob fi rous scao ff lds in nerve injured sites may inhibit the apoptosis of neurons. e diff Th erentiation of PC12 cells induced by NGF was also improved on LBP loaded nanob fi ers, as evidenced by morphological evaluation and immunouo fl rescence staining (Fig. 5). Although the mechanism of LBP in PC12 cells differentiation was not known exactly, our results revealed that the differentiation of PC12 cells induced by NGF could be promoted by LBP to a certain extent. Previous studies by Ho et al. reported that LBP could reduce the cell death of Rat primary cultured cortical neurons treated with homocysteine or glutamate, and the possible Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 9 www.nature.com/scientificreports/ Figure 8. (a) Fluorescence images of DRG neurons cultured on PLGA-LBP0 and PLGA-LBP30 scao ff lds for 7 days (Green: NF160 stained with Alexa Fluor 488-conjugated goat anti-mouse lgG H&L; Blue: nuclei counterstained with DAPI). (b) Neurite length of DRG neurons on PLGA-LBP0 and PLGA-LBP30 scao ff lds aer 7 d ft ays culture (Data were mean ± SD, n = 10; *denotes significance between two groups using one-way ANOVA test; P < 0.05). 21,43 mechanism was to up regulating the phosphorylation of Extracellular Regulated Kinase (ERK) . It has been known that the process of phosphorylation of ERK is the necessary requirement of PC12 cells differentiation 44–46 induced by NGF . u Th s, we supposed that the LBP could facilitate the PC12 cells differentiation induced by NGF through the up regulation of phosphorylation of ERK. Additionally, the present study found that the expression of MBP in Schwann cells showed significant enhancement on PLGA-LBP30 group (Fig. 7). Gao et 24 2+ al. reported that LBP could decrease the level of intracellular Ca , while some research have shown that intra- 2+ cellular Ca level appears to ae ff ct cell viability, proliferation and differentiation in Schwann cells and neuronal 47–49 cells . Therefore, we considered that the Schwann cells myelination was improved by LBP through the decrease 2+ 2+ of intracellular Ca . Then the decrease of intracellular Ca level may lead to inactivation of protein kinase C (PKC), which is a key protein kinase enzyme involved in other signal pathways such as mitogen-activated protein 50,51 kinase (MAPK) to indirectly influence Schwann cells myelination . Rat DRG neurons showed better viabil- ity and neurite out growth on LBP containing nanofibers, which could be seen in Fig. 8. We surmised that the synergistic ee ff ct of NGF and LBP was more ee ff ctive than the independent induction of NGF in promoting the axon growth. In brief, the existence of LBP showed a remarkable increase in the neurite outgrowth of the rat DRG neurons and up-regulation of the NF160 expression, although the synergistic reaction mechanism between NGF and LBP was not known completely. Taken together, these results demonstrated that LBP has a positive effect on nerve cells proliferation and differentiation in vitro. There was a beneficial synergistic effect between NGF and LBP in promoting PC12 cells differentiation and axonal extension of neurons. To allow regeneration following peripheral nerve injury, the microenvironment must be permissive enough for neurite outgrowth. Therefore, the LBP loaded nanofibrous scao ff lds, especially PLGA-LBP30, could be a promising substrate for target peripheral nerve injury repair. Materials and Methods Materials. e 75/25 P Th oly (lactic-co-glycolic acid) (PLGA) (inherent viscosity 0.61 dL/g) was purchased from Jinan Daigang Biomaterial CO., Ltd, China. Lycium barbarum polysaccharide was provided by Guangdong- Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University. 1,1,1,3,3,3-Hexauo fl ro-2-propanol (HFP), glutaraldehyde, Dulbecco’s modified eagle’s medium (DMEM/F12), were purchased from Sigma, Singapore. Nerve growth factor (NGF) was purchased from Millipore, Singapore. Rat pheochromocytoma (PC12) cells in the Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 10 www.nature.com/scientificreports/ adherent type and Schwann cells were obtained from ATCC, USA, while fetal bovine serum (FBS), horse serum (HS) and trypsin/EDTA were purchased from GIBCO Invitrogen, USA. Alamar Blue (AbD Serotec) was purchased from Chemoscience, Singapore. Schwann cell medium was purchase from Gene-Ethics Asia Pte Ltd, Singapore. Fabrication of scaffolds. 28% w/v PLGA solution was prepared by dissolving PLGA into HFP followed by a magnetic stirring at room temperature overnight. Meanwhile, LBP aqueous solution with 10 mg/ml, 30 mg/ml, and 50 mg/ml concentration were prepared separately. Core-shell structured PLGA-LBP nanofibers were fabri- cated via our lab’s electrospinning set-up as shown in Fig. 1a. The PLGA solution and LBP aqueous solution were separately placed into 5 ml plastic syringes and fed through a compound nozzle with an inner needle coaxially placed inside an outer one at a rate of 1 ml/h and 0.2 ml/h using two syringe pump respectively. The exit orifice diameters of the inner and outer needles were 0.5 and 0.8 mm. e Th high voltage applied during electrospinning was 18 kV, while the distance between the tip of the needle and grounded steel plate covered with aluminum foil was set at 13 cm. The nanofibrous scaffolds fabricated with LBP aqueous solution of 10 mg/ml, 30 mg/ml, and 50 mg/ml concentration were separately referred as PLGA-LBP10, PLGA-LBP30, and PLGA-LBP50. Pure PLGA scao ff lds were also prepared as control by conventional electrospinning, and labeled as PLGA-LBP0. Characterization of scaffolds. e Th surface morphology of nanobr fi ous scaffolds . e Th scanning electron micro- scope (SEM) was applied to observe the surface topography of fabricated nanob fi rous scao ff lds. e Th electrospun nano- b fi ers were sputter-coated with gold (JEOL JFC-1200 Fine Coater, Tokyo, Japan) and then visualized using a SEM (Model S-4300, Hitachi, Tokyo, Japan). The average diameter of the nanob fi ers was calculated from 100 random points chosen from the SEM images with analysis by Image J sowa ft re (National Institute of Health, Bethesda, MD). The core-shell structure of nanob fi rous scao ff lds. The core-shell structure of the nanofibers was verified using transmission electronic microscopy (JEM-3010, JEOL, Tokyo, Japan) at 200 kV. The samples for TEM observation were prepared by collecting nanofibers with carbon-coated copper grids. Laser scanning confocal microscopy (LSCM, Zeiss LSM700) was used to further confirm the distribution of LBP in the core of nanob fi ers, and to do so, the LBP aqueous solution used for electrospinning was mixed with FITC. e m Th echanical property of nanob fi rous scao ff lds. Tensile properties of the nanob fi rous scao ff lds were evaluated using a universal materials tester (H5K-S, Hounsfield, UK) at an ambient temperature of 20 °C and humidity of 65%. Tests were performed on rectangular specimens with dimensions of 30 × 10 mm. A cross-head speed of −1 10 mm min was applied for all specimens during the process. Six specimens of each sample were measured. e m Th easurement of water contact angle. Hydrophilicity of the electrospun nanob fi rous scao ff lds was measured by water contact angle measurement using VCA Optima Surface Analysis System (AST products, Billerica, MA, USA). To prepare samples for water contact angle measurement, the nanob fi ers were collected on coverslips and placed on the testing plate. e Th n deionized water was dropped on the specimens and the data was recorded. Three different points of the same specimen were measured and each sample was performed in triplicate. Release of LBP from core-shell nanofibers in vitro. PLGA-LBP30 nanofibrous scaffold was used to evaluate the release behavior of LBP in vitro. 10 mg of PLGA-LBP30 nanob fi rous scao ff lds were soaked in a 2 ml centrifuge tube with 1 ml of PBS. Subsequently, the centrifuge tubes with nanofibrous mats were all incubated in a continuous horizontal shaker at a speed of 150 rpm at 37 °C. At preset time points, the PBS was retrieved completely from the tubes and the equal volume of fresh PBS was replaced for continuous incubation. The LBP concentration was determined by UV-vis spectrophotometer at wavelength at 260 nm. The cumulative amount of released LBP was calculated. The samples for each time point were run in triplicate. Degradation of nanofibrous scaffolds in vitro. The degradation of nanofibrous scaffolds were per - formed as described previously . Briefly, the nanob fi er meshes were cut into small square pieces. Then the spec- imens were immersed in 10 ml of PBS in 15 ml centrifuge tubes. Subsequently, the tubes were kept in a shaking incubator at 37 °C operating at 150 rpm. At each particular time point of study, the specimens were taken out from the tubes, rinsed several times with distilled water to remove residual buffer salts, and dried in a vacuum desiccator. The morphology was observed with SEM as described previously. Cell culture in vitro. PC12 cell culture. PC12 cells were cultured in normal Growth Medium composed of Dulbecco’s modified eagle’s medium (DMEM/F12) supplemented with 10% horse serum (HS), 5% fetal bovine serum (FBS), and 1% antibiotic/antimycotic solution in a 75 cm cell culture flask. Cells were maintained at 37 °C in a humid- ified incubator with 5% CO , and the culture medium was changed once in every two days. The nanob fi rous scao ff lds for cell culture were collected with 15 mm cover slips. All the samples were sterilized for 5 h under UV light in biological safety cabinet (Bioclassic Class II Series Type A2, Gelman Laminar Airflow Systems) and then placed in a 24-well plate pressed with stainless steel rings. Subsequently all samples were washed thrice with PBS, and immersed in Growth Medium until cell seeded. Aer ft the confluence was sufficient, PC12 cells in a fl sk were detached by trypsin/EDTA, counted by automated cell counter, and seeded on all nanob fi rous scao ff lds with a density of 1.0 × 10 cells/well. Schwann cell culture. Similarly, the Rat Schwann cells were grown in SCM medium with 10% FBS. And before seeding the cells, the scao ff lds need coating with Poly-L-lysine. In detail, the scao ff lds were washed with PBS for three times aer s ft terilization under UV light, and then incubated with 200 μl Poly-L-lysine solution (Sigma, Singapore) for 2 h in 37 °C incubator. Subsequently, the scao ff lds were washed again with PBS for three times, and placed in an incubator overnight to allow the scao ff lds to be dry fully. All the other followed process was the same as PC12 cells. Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 11 www.nature.com/scientificreports/ Cell behavior of PC12 cells on scaffolds. e Th proliferation of PC12 cells. e p Th roliferation of PC12 on different scaffolds was performed after 1, 3, 5, 7 days of culture by means of the Alamar Blue assay. At pre- determined time points, medium was carefully removed and 1 ml of Alamar Blue solution (10% Alamar Blue, 90% DMED/F12 medium) was added to each well followed by incubation in darkness for 4 h under 37 °C. Aer ft incubation, the solution was pipetted from each well into 96-well plate and read at 560 nm (excitation)/590 nm (emission) by a Varioskan flash reader (Thermo Fisher Scientific, MA, USA). Neuronal dier ff entiation of PC12 cells. For differentiation study of PC12 cells, the medium was changed from +NGF Growth Medium to Differentiation Medium composed of DMED/F12 supplemented with 1% HS, 0.5% FBS, −1 1% antibiotic/antimitotic solution and 100 ng ml NGF aer ce ft lls seeded for 1 day. For comparison, cells were −NGF also cultured with the same medium above without NGF which is referred as Differentiation Medium . The morphology of differentiated PC12 cells was different from its original ones. To evaluate the neuronal differentiation of PC12 cells, the morphological changes were observed with SEM aer 10 d ft ays of incubation in Differentiation Medium. Meanwhile, the morphology of cells cultured on scao ff lds with Growth Medium was also observed as control. Aer 10 d ft ays incubation, cells-scao ff lds constructs were rinsed twice with PBS and then fixed with formalin for 20 min. Aer t ft hat, all samples were rinsed thrice with PBS and dehydrated with a series of graded ethanol (50%, 70%, 90% and 100%) for 15 min each, followed by air-drying overnight in room tempera- ture. Finally, the dry samples were sputter coated with gold and observed under SEM. The neuronal differentiation of PC12 cells was also evaluated by immunostaining of neuronal specific protein-neurofilament 200 (NF200). After 10 days of cell culture in Differentiation Medium, all scao ff lds were rinsed with PBS, fixed in formalin for 20 min and permeabilized with 0.1% Triton-X100 for 5 min. Then 3% BSA solution was used to block nonspecific binding for 90 min. After that, the samples were stained with primary antibody, anti-NF200 produced in rabbit (Sigma) at a dilution of 1:100 for 2 h at room temperature. Subsequently, all samples were washed thrice with PBS and stained with secondary antibody, FITC conjugated goat anti rabbit (Sigma) at a dilution of 1:300 for 1 h. Aer r ft insing with PBS for five times, the scao ff lds were treated with mount- ing medium with DAPI (Vector Laboratories, USA). Finally, the immunostained samples were visualized under LSCM to observe the cell phenotype and neurite extension of differentiated PC12 cells. Cell behavior of Schwann cells and DRG neurons on scaffolds. The proliferation of Schwann cells. The proliferation of Schwann cells on different scao ff lds was performed aer 1, 3, 5, 7 d ft ays of culture by means of the Alamar Blue assay. The detailed process was the same as what described in section of the proliferation of PC12 cells. Cell morphology study of Schwann cells. e Th morphology of Schwann cells on different scao ff lds was observed by both SEM and fluorescent microscopy. For SEM observation, all the process was the same as what described in section of Neuronal dier ff entiation of PC12 cells. For the fluorescent microscopy observation, immunostaining of specific protein for Schwann cells, namely S100, was carried out. And the followed process was similar with that described in section of Neuronal dier ff entiation of PC12 cells. The primary antibody used in this study was rabbit anti-S100 (diluted at 1:200, Sigma). Myelin basic protein (MBP) expression of Schwann cells. Myelination of Schwann cells is critical for the success of peripheral nerve regeneration. To evaluate the inue fl nce of PLGA-LBP nanofibrous scao ff ld on modulating myelination of Schwann cells, the expression of MBP of Schwann cell was determined with immunostaining. The process of immunostaining was the same as what described in section of Neuronal dier ff entiation of PC12 cells. Anti-MBP antibody produced in rabbit (diluted at 1:200, Sigma) was used as the primary antibody in this study. DRG neurons outgrowth on nanob fi rous scao ff lds. DRG neurons were isolated from the embryo of the 14 day pregnant rat. Before seeding the DRG neurons, the samples were exposed to UV light overnight for sterilization, followed by thrice rinse with PBS. The culture medium for DRG neurons was Neuralbasal medium with 1% −1 Penicillin-Streptomycin Solution, 2% B27 supplement factors and 20 ng ml NGF. The culture medium with NGF was replaced every two days. After 7 days incubation, the DRG neurons were fixed for 30 min in a 4% paraformaldehyde at 4 °C and then washed three times with PBS. Subsequently, the DRGs were immunostained with the primary antibody solution overnight at 4 °C. Aer wa ft shing with PBS for three times, the samples were incubated with Goat anti-rabbit IgG Alexa 488 (Invitrogen, Camarillo, CA, USA) diluted 1:200 in PBS as the secondary antibody. 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Acknowledgements i Th s study was funded by Leading Talents of Guangdong (2103), Programme of Introducing Talents of Discipline to Universities (B14063) and Ningxia Key Research and Development Program Grant. Author Contributions Jing Wang, Lingling Tian and Seeram Ramakrishna conceived of the study and participated in its design and coordination. Jing Wang and Nuan Chen performed the experiments. Jing Wang and Lingling Tian drae ft d the main manuscript and performed the statistical analysis. Liumin He, Kwok-Fai So and Xiumei Mo corrected the original draft. All authors read and approved the final manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26837-z. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific RePo R Ts | (2018) 8:8669 | DOI:10.1038/s41598-018-26837-z 14
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