Annals of Biomedical Engineering ( 2015) DOI: 10.1007/s10439-015-1311-x
Potential Neurogenesis of Human Adipose-Derived Stem Cells on Electrospun Catalpol-Loaded Composite Nanofibrous Scaffolds JIAN-HUI GUO,1,2 YANG LIU,1,2 ZHENG-JUN LV,3 WEN-JUAN WEI,1 XIN GUAN,1 QING-LIN GUAN,4 ZHI-QIAN LENG,1 JING-YUAN ZHAO,1 HUI MIAO,1 and JING LIU1,2 1 Regenerative Medicine Center, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, People’s Republic of China; 2Institute of Integrative Medicine, Dalian Medical University, Dalian 116044, People’s Republic of China; 3Queen Mary University of London, London E1 4NS, UK; and 4Center Laboratory, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, People’s Republic of China
(Received 18 October 2014; accepted 23 March 2015) Associate Editor Peter E. McHugh oversaw the review of this article.
Abstract—Catalpol, a natural active ingredient extracted from the traditional Chinese medicine, was verified exhibiting beneficial effects on neural differentiation compared with commonly used chemical inducers by our previous studies. The aim of this study was to evaluate the effects of catalpolloaded scaffold on guiding neuronal differentiation of human adipose tissue-derived stem cells (hASCs). Fabrication technique of catalpol loading into the electrospun poly(lactic-coglycolic acid)/multi-walled carbon nanotubes/silk fibroin nanofibrous scaffolds was successfully established. The topographical and mechanical properties of the nanofibers scaffolds were characterized by scanning electron microscopy and tensile instrument, respectively. In vitro catalpol release was studied in phosphate-buffered solution at 37 C. Immunnocytochemistry, RT-PCR, and western blot assays were performed to estimate hASCs neuronal differentiation, and it was shown that catalpol has significantly upregulated the expressions of bIII-tubulin and Nissl. Our experiments demonstrated that catalpol, as a traditional Chinese medicine extract, could be encapsulated into composite nanofibers and induce differentiation of hASCs into neural-like cells, which might offer new avenues in nerve regeneration. Keywords—Electrospinning, Nanofibrous, Catalpol, Neurogenesis, Adipose tissue-derived stem cells.
INTRODUCTION Nerve injuries often result in severe damage since neural tissue possesses very limited capacity to regenerate new functional neurons. Tissue engineered neural Address correspondence to Jing Liu, Institute of Integrative Medicine, Dalian Medical University, Dalian 116044, People’s Republic of China. Electronic mail: [email protected] Jian-Hui Guo and Yang Liu have contributed equally to this work.
tissues may serve as a promising alternative for neural regeneration. Currently, clinical studies are focused on the delivery of stem cells capable of differentiation into neurons.5,8,9 However, such stem cells would need to proliferate and differentiate into the desired phenotype with the aid of adequate chemical, mechanical, or biological stimuli.28 Certain biomolecules, such as growth factors, genes, or drugs, can be incorporated within tissue engineering scaffolds to promote the proliferation and neuronal differentiation of the seeded cells, thereby enhancing nerve tissue regeneration.14 The electrospinning technique offers a possibility of incorporating protein growth factors or drugs within polymer nanofibers, which could then serve as a source of continued and controlled release of the growth factor or drugs.28 Electrospinning, a simple and versatile technique for preparing nanofibrous nonwoven mats, has been successfully developed to mimic the architecture of endogenous extracellular matrix (ECM). Various studies have confirmed that electrospun nanofibrous scaffolds could significantly facilitate cell adhesion and proliferation, drug loading, nutrients delivery, and waste removal due to their high porosity, spatial interconnectivity, and surface/volume ratio.12,23,30 The nanofibrous structures could also induce the extension of neurons, which are beneficial for during nerve regeneration.19,20 The electrospun nanofibers based on a wide range of biodegradable and biocompatible materials including natural proteins and synthetic polymers has been successfully prepared for tissue engineering applications. Among various synthetic polymers, poly(lactic-co-glycolic acid) (PLGA), approved by the US Food and Drug Administration for 2015 Biomedical Engineering Society
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several biomedical applications, has been widely used in tissue engineering.18 Silk fibroin (SF), one of strongest and toughest biological material, has become a promising biomaterial for tissue engineering and drug carrier due to its biocompatibility and the ability to control the release of growth factors.24 Carbon nanotubes (CNTs) are a conductive biomaterial with sizes comparable to ECM molecules such as collagens and laminins, which have been reported to favor neuronal growth.3 Both PLGA and SF could be fabricated into nanofibrous structures via electrospinning process, providing improved microenvironment for cell adhesion and proliferation. However, further improvement is still required when silk and PLGA are used in nerve tissue regeneration, where blending with conductive multi-walled carbon nanotubes (MWCNTs) in electrospun nanofibers might be an effective way. Adipose tissue is emerging as a source of stem cells obtained by less invasive methods such as lipoaspiration, and in larger quantities than bone marrow. Adipose tissue-derived stem cells (ASCs) have been found to have the potential in differentiating into functional neuronal-like cells.13 All of these advantages have made ASCs to be ideal cells for the treatment of neurological diseases. Catalpol, an iridoid glycoside isolated from the roots of traditional Chinese medical herb rehmannia, has been widely used as a traditional Chinese medicine for the treatment of stroke and diseases of aging owing to its neuroprotective effects. It has been reported that catalpol can induce neuronal differentiation in PC12 cells through activation of the intracellular signal transduction pathway, and promote neurite length.36 Our previous studies further confirmed that catalpol could improve neuronal differentiation and cell viability of human ASCs in vitro under two-dimensional condition, indicating promising future of catalpol as a neurogenesis inducer (unpublished data). Therefore, incorporation of catalpol into a composite nanofibrous scaffolds may improve their clinical efficacy in nerve tissue engineering. In our present study, catalpol was loaded into PLGA/MWCNTs/SF electrospun composite to directly induce hASCs into neuronal-like cells. The morphology, diameter distribution, and porosity of the fibers were studied. In addition, the mechanical properties of the composite nanofibrous scaffolds were characterized at dry and wet state, respectively. The release behaviors of catalpol and its influence on neuronal differentiation of hASCs were studied to assess the feasibility of the composite as neural regeneration matrice. To our knowledge, this is the first report as regard to the neurogenesis effects study of the electrospun catalpol-loaded composite nanofiber scaffolds.
MATERIALS AND METHODS Chemicals PLGA (85:15, Mw = 100,000) was obtained from Daigang Biomaterials Company (Jinan, China). 1,1,1,3,3,3-Hexafluor-2-propanol (HFP) was purchased from Xiya Reagent Company (Chengdu, China). Silk fibroin (SF) was obtained from Chinatiansi Biotek Company (Huzhou, China). MWCNTs were purchased from Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences (Chengdu, China); Catalpol (purity > 99%) was obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Fabrication of PLGA/MWCNTs/SF/Catalpol Nanofibers PLGA/MWCNTs/SF/catalpol composite nanofibers were fabricated using the electrospinning technique. The composite PLGA/MWCNTs/SF suspensions with catalpol at various concentrations were obtained by mixing the PLGA, MWCNTs, SF and catalpol solutions together (3:1:2:1, volume ratio). Firstly, PLGA was dissolved in HFP and incubated in 37 C water bath overnight. SF was added into HFP and stirred continuously for 60 min at room temperature. Meanwhile, MWCNTs and catalpol were dissolved in HFP under ultra-sonication for 60 and 10 min, respectively. Finally, the suspensions were stirred continuously for 30 min before electrospinning. Herein, the concentrations of PLGA, MWCNTs, and SF in the electrospinning solutions were fixed at 15, 0.2 and 10% (w/v), respectively. And the concentrations of catalpol were 0, 1, 2, 3, 4, and 5 mg/mL, respectively. The scaffolds without catalpol were set as the control group. The polymer solutions were then fed into a 10 mL syringe (BD, China) coupled with to a 9 G stainless steel needle using a syringe pump (LongerPump, China) at a flow rate of 1.0 mL/h. High voltage (Teslaman, China) of 15 kV was applied, and the fibers were collected on an aluminum foil wrapped collector kept at a distance of 10 cm from the needle tip. Collected nanofibers were dried overnight and used for the characterization and cell culture experiments. Morphology and Characterization of Electrospun Nanofibers Scanning Electron Microscopy The morphology of electrospun nanofibers was observed under scanning electron microscope (SEM) (JEOL-JSM 6360-LV, Japan) at an accelerating voltage
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of 20 kV, after coating with a thin gold layer. For local high magnification, another SEM (SUPRA 55, Zeiss, Germany) was also used. The average diameter of the fibers was analyzed by an image analysis software (Image J, National Institute of Health, USA) based on the SEM images. At least 200 nanofibers from different images were determined for each sample. Surface Porosity Determination The surface porosity was calculated based on SEM images.1 Briefly, the porosity was determined by calculating volume beneath the surface and volume beneath the same surface. SEM images were firstly converted into two-dimensional matrix in gray scale format and it was assumed that the height was equal to maximum height of any point on the sample’s surface. Then the surface porosity calculation was processed by OriginPro software (OriginLab Corp, USA). Mechanical Property Study Tensile properties of electrospun nanofibers in the dry and wet state were determined using a tensile tester (Instron 5989, Canton, USA) at room temperature. The specimens were cut into rectangular pieces with the dimensions of 50 mm 9 10 mm, and the gage length of 10 mm. The thickness of each specimen was measured with a micrometer before testing and the distance between the gripping points was 30 mm. The mechanical testing was conducted with the grips moving at a stretch speed of 20 mm/min. A minimum of five strips from different sites of each sample were tested, and Young’s modulus, yield strength, and elongation at break were calculated. For the measurement in the wet state, the samples were soaked in phosphate-buffered solution (PBS) at 37 C for 1 h and then measured as described above. In Vitro Release of Catalpol from Nanofibers The electrospun samples were cut into square pieces of 25 mm 9 25 mm, and their weights were accurately measured. Then the samples were placed in centrifuge tubes containing 10 mL phosphate-buffered saline (PBS, pH 7.4), and were incubated in a 37 C water bath with constant sharking rate at 60 cycles/min for a period of 7 days. 1 mL solution was taken out from three tubes (triplicates) and 1 mL fresh PBS was added back at scheduled time points. The collected solutions were stored at 220 C, and thawed at room temperature before analysis. After filtration through a 0.22 lm membrane (Millipore, USA), samples were analyzed using high performance liquid chromatography (HPLC) to determine the amount of catalpol released as a function of time. The column type was
Hypersil C18 (5 lm of 200 mm 9 4.6 mm internal diameter). The solvent for calibration was 0.5 mg/mL catalpol in the solvent of acetonitrile and water (0.5:99.5, v/v), k = 210 nm. The percentage of the detectable catalpol release was determined using a preestablished standard curve based on the initial weight of catalpol incorporated in the nanofibers. Standard curve was constructed in the range 1.0–500.0 lg/mL for catalpol. The regression equation was calculated and the coefficient was determined (r2 = 0.9997). Human ASCs Culture and Seeding on Scaffolds Human ASCs were isolated and cultured as mentioned in the previous method.10 Briefly, adipose tissue was obtained from human liposuction surgery with informed consent, under the requirements of Ethic Committee of the First Affiliated Hospital of Dalian Medical University. The adipose tissues were washed extensively with PBS and then minced into 1 mm3 pieces. The adipose tissues were digested with collagenase I at 37 C for 60 min, and neutralized by adding human Adipose-derived Stem Cell Growth Medium (OriCellTM, Cyagen Biosciences, China). The digested adipose tissues were filtered with a nylon membrane (Fisher, USA) to remove undigested fragments and then the cellular suspension was centrifuged at 1000 rpm for 10 min. After three washes with PBS (centrifuged at 1000 rpm, 5 min), cells were incubated at 37 C in a humidified atmosphere containing 5% CO2, and medium was changed every 3 days. hASCs of passage 4–6 were used in the following experiments. Before cell seeding, the electrospun PLGA/ MWCNTs/SF/catalpol scaffolds were sterilized under ultraviolet light for 30 min on each side, and washed twice with PBS for 5 min each to remove residual solvent. Then the fibers were subsequently immersed in High-glucose Dulbecco’s Modified Eagle’s Medium (H-DMEM) (Invitrogen, USA) overnight before cell seeding. hASCs grown in 25 cm2 cell culture flasks (Corning, USA) were detached by 0.25% trypsin– 0.1% ethylene diaminotetraacetic acid (EDTA) (Invitrogen, USA). A density of 5000 cells/well were seeded on the scaffold in a 24-well plate (Corning, USA) and cultured in H-DMEM supplemented with 1% penicillin–streptomycin (Invitrogen, USA; termed as ‘‘hASC growth media’’). Cells seeded on tissue culture polystyrene (TCP) in 24-well plates cultured in Adipose-derived Stem Cell Growth Medium at the same density were served as negative control. Immunocytochemistry for hASCs Neurogenesis After 3 and 5 days culture on electrospun PLGA/ MWCNTs/SF/catalpol nanofibers in ‘‘hASC growth
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media’’, immunocytochemistry for hASCs neurogenesis was performed. The cells were initially fixed with 4% paraformaldehyde for 30 min at room temperature, and then permeabilized with 0.1% Triton X100 for 10 min and washed three times with PBS. Nonspecific antibody binding was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and then the cells were exposed to rabbit antiIII-Tubulin (1:1000) overnight at 4 C. After washed for three times with PBS, the cells were incubated with goat anti-rabbit fluorescein isothiocyanate secondary antibody (FITC, 1:1000) (all from Sigma, USA) for 1 h at room temperature, and then washed with PBS again. Finally, the samples were stained with Nissl-PE (1:200, Molecular, USA) for 15 min, and the nuclei were then stained with Hoechst 33258 (Sigma, USA) for 15 min, followed by three washes with PBS. Fluorescence images were taken by a laser scanning confocal microscope (Leica SP8, Germany). At least ten images were chosen randomly from each staining, and the number of positive cells were calculated and analyzed.
Real-Time Polymerase Chain Reaction (RT-PCR) Analysis RNA was isolated using the RNAiso Plus (Takara, Japan). RNA concentration was measured using the NanoDrop 2000 (Thermo, USA). Total RNA (1 mg) was subjected to reverse transcription (RT) by PrimeScript RT reagent Kit (37 C for 15 min, 85 C for 5 s) (Takara, Japan). Primers were designed by PRIMER EXPRESS software (PE Applied Biosystems, USA). Quantitative real-time PCR was performed using Light-Cycler 480 (Roche Applied Science, USA) with SYBR Green I (Takara, Japan) as the detection system. After denaturing for 30 s at 95 C, amplification was performed with 40 cycles of 5 s at 95 C and 20 s at 56 C. Melting curve analysis was performed with 1 s at 95 C, 15 s at 65 C, and 1 s at 95 C, melting curve analysis was used to confirm the specificity of amplification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a standard control for the normalization of the gene expression level, and the gene expression levels of the cells on the TCP were used as a negative control group. Results were analyzed by the value of DDCT using Light-Cycler software (Roche, USA). The sequences of PCR primers (forward and backward, 5¢–3¢) were as follows: bIII-tubulin: forward 5¢-GAACCCGGAACCAT GGACAG-3¢ reverse 5¢-GACCCTTGGCCCAGT TGTTG-3¢
GAPDH: forward 5¢-AACGGATTTGGTCGTA TTGGG-3¢ reverse 5¢-TGGAAGATGGTGATGG GATTT-3¢
Western Blot Analysis hASCs were removed from nanofibrous composite scaffolds, and lysed with RIPA buffer. Protein concentration was measured using BCA protein kit. Extracts were re-suspended with 59 SDS-polyacrylamide (PAGE) gel buffer (all from Beyotime Biotech, China) and boiled for 7–8 min. Equal amounts of protein extracts (20–30 mg) were fractioned on a 10% SDSPAGE. Following electrophoresis, the proteins were transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, USA). After the membrane was blocked with blocking buffer (0.1% Tween 20, and 5% skim milk in PBS, pH 7.4) for 1 h, the membrane was incubated overnight at 4 C with anti-bIII-tubulin (Sigma, USA) diluted 1:1500 in TBST (Tris-buffered saline-Tween 20) and anti-GAPDH primary antibodies at 4 C for overnight. After washing three times with TBST, the membrane was incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (diluted 1:1000 in TBST, Abcam, USA). After washing the membrane, labeled proteins were visualized with enhanced chemiluminescence (ECL) detection reagents (Amersham Pharmacia Biotech., USA) and were analyzed using an imaging analysis system (Gelpro32, MediaCybernetics, USA). Statistical Analysis Experiments were performed with triplicate samples and all data were presented as mean ± standard deviation (SD). Single factor analysis of variance (ANOVA) was performed to determine statistical significance and Fisher’s LSD for multiple comparisons. The statistical software package SPSS 13.0 (SPSS, Chicago, IL, USA) was used for the data analysis. A value of p < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION Characterization of the Composite Electrospun Nanofibrous Scaffolds PLGA/MWCNTs/SF/catalpol nanofibers fabricated by electrospinning showed nonwoven mat structures and random fiber distribution with smooth and beadless fibers morphology, as shown in Fig. 1. Higher magnification SEM images (A3–F3, Fig. 1) revealed
Neurogenesis of hASCs on Catalpol-Loaded Nanofibers
that the nanofibers were with smooth surface and no catalpol particles on the fiber surfaces, indicating that catalpol distributed uniformly in nanofibers and verifying PLGA/MWCNTs/SF/catalpol combination is a reliable and reproducible electrospinning system. The diameter distribution of the fibers (Fig. 1A4–F4) showed diameter of nanofibers decreased nonlinearly with the increase of catalpol concentration. The diameters of the electrospun nanofibers have an influence
on cell behavior, which are dependent on the electrospinning conditions, such as concentration of the polymer solutions, voltage, flow rates and the collecting distance. Christopherson et al. have investigated the influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation and demonstrated that as the fiber diameter decreased, higher degree of proliferation and cell spreading were observed.7 In this study, incorporation of catalopl
FIGURE 1. Morphology characterization of composite electrospun nanofibers by SEM. Nanofibrous scaffolds were electrospun at distance of 10 cm and flow rate of 1.0 mL/h. Catalpol concentration was 0, 1, 2, 3, 4, and 5 mg/mL, respectively. PLGA/MWCNTs/ SF catalpol-loaded scaffolds exhibited fairly uniform fibers without the presence of beads along the fibers. A1–F1: low magnification (31000); A2–F2: 310,000; A3–F3: high magnification (3200,000); A4–F4 showed the diameter distribution histogram of the composite nanofibers.
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FIGURE 2. The porosity of nanofibers analyzed by Image J software from SEM iamges. **, and *** represents p < 0.01, and p < 0.001, compared with control group, respectively.
decreased the diameter of nanofibers. The explanation may be that addition of small molecules, such as catalpol, could increase the concentration of charged particles, thus raise the electric force of the polymer solution, and facilitate decreased fiber diameter. Zeng et al. reported that the diameters of the fibers were sharply decreased, since the addition of drugs (such as rifampin) had reduced the surface tension,38 which was consistent with our results. A perfect three-dimensional nanofibrous scaffold can afford not only cell attachment, proliferation, and differentiation, but also sufficient transport for nutrients and waste removal.21 Nanofibrous scaffolds with high porosity can promote cells growth and provide a three-dimensional structure similar to native tissue. The porosity of PLGA/MWCNTs/SF nanofibers with different concentrations of catalpol was calculated according to the method demonstrated by Abdullah and Khairurrijal.1 As demonstrated in Fig. 2, the porosities of PLGA/MWCNTs/SF nanofibers with 3 mg/mL and 4 mg/mL of catalpol were significantly higher than those in control group. The porosities of nanofibers were 60.35 ± 0.23, 61.61 ± 1.46, and 50.67 ± 1.83%, respectively, for nanofibers with 3 mg/ mL, 4 mg/mL catalpol, and the control group. However, there was no significant difference in nanofiber porosities between control group and the other experimental groups. Our results showed that catalpol has maintained the high porosity of the composite scaffolds. The values of Young’s modulus, yield strength, and elongation at break of the samples were determined and illustrated in Fig. 3. We expect that electrospun PLGA/MWCNTs/SF composite nanofibers could display excellent mechanical properties. Results of mechanical evaluations showed that the Young’s modulus of nanofibrous scaffolds were greatly im-
FIGURE 3. Mechanical properties of electrospun composite scaffolds with different concentrations of catalpol at dry and wet state. (a) Young’s modulus; (b) yield strength; (c) elongation at break. *, **, and *** represents p < 0.05, p < 0.01, and p < 0.001, compared with control group, respectively. All values are represented as mean 6 SD (n 5 5).
proved with the introduction of catalpol, thus creating a biocomposite scaffold with superior tensile properties, which was especially suitable for nerve tissue engineering (Fig. 3a). There were no significant differences among groups with regards to yield strength when the scaffolds were in dry state (Fig. 3b). The elongation at break of nanofibers containing catalpol
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was significantly higher than that of control group, except for the 1 mg/mL catalpol group (Fig. 3c). With the introduction of catalpol, the hydrophilicity of PLGA/MWCNTs/SF membrane was increased, causing the decrease of Young’s modulus in wet state. After incubation in PBS, the electrospun composite membrane exhibited a significant increase in both of yield strength and elongation at break with respect to those in dry state. Mechanical properties of nanofibrous scaffolds are essential for tissue engineering. In particular, appropriate mechanical properties can help with providing support and guidance for axons regeneration. The ideal scaffolds are ones with mechanical properties close to in vivo tissues, and this is the reason we chose MWCNTs as a composition in the polymer solution, aiming to enhance the overall mechanical properties of the scaffolds.
FIGURE 4. In vitro release profile of catalpol from electrospun composite scaffolds with different concentrations of catalpol. The scaffolds were incubated in PBS (pH 7.4) at 37 °C, and the samples were analyzed with HPLC to determine the amount of catalpol released as a function of time. ** represents p < 0.01 compared with the other groups.
In Vitro Catalpol Release Incorporation of growth factors,16 vitamins,30 or antibiotics into biocompatible and biodegradable nanofibers might be a promising alternative to regulate cell proliferation and differentiation. Catalpol has exhibited various biological activities, including antioxidation, anti-apoptosis, and radio-protection,4 and was loaded in electrospun nanofibers to regulate cell behaviors in our study. The controlled release of catalpol from the composite nanofibrous scaffolds was analyzed by HPLC, and their release profiles from electrospun nanofibers at a temperature of 37 C and pH 7.4 were shown in Fig. 4. The release profile of catalpol had three stages: an initial burst release, further a constant stage, followed by the decrease. The catalpol released quite fast during the initial 1 h, and then maintained a slight decreasing stage from 1 to 24 h. After 24 h, the release rate slowed down until no catalpol could be detected at day 7. As shown in Fig. 4, the highest detectable drug release was over 51.20 ± 5.65%, which appeared in the nanofibrous scaffolds loaded with 5 mg/mL. The initial release percentages of nanofibers with different concentrations of catalpol at 1 h were around 50%, the lowest value was 48.61 ± 5.36% of nanofibers with 3 mg/mL catalpol. From 1 to 12 h, release percentages decreased in all groups, and on the contrary, during 12–24 h, the release rate remained stable in each group. The detectable catalpol release of electrospun nanofibers with 1 mg/mL catalpol was significantly lower than that of the other groups at 4, 12, and 24 h. From 24 h to 5 days, all groups sustained downward trends to the lowest point, with the lowest value of 9.38 ± 2.88 and 19.18 ± 4.41% for 1 and 5 mg/mL catalpol group, respectively. It is obvious that the ini-
tial releases were all above 50%, which is the common problem caused by blending, namely burst release. The initial burst release results in hard control of sustained drug release, therefore it is necessary to avoid burst release;15 however, it may be useful for drug release system which requires a large initial dose and long-term maintenance of the drug. Researcher are making efforts to avoid burst release using the methods of shell–core electrospinning,15 mixing the drug with the polymer by chemical binding,26 multilayered continuous electrospinning.11 Zeng et al. have explained that burst release phenomenon occurred due to the differences in dissolution characteristics of the drug and its associated polymer.38 In their work, paclitaxel, doxorubicin base were lipid-soluble drugs, while doxorubicin hydrochloride was water-soluble drug. When the water-soluble drug was blended with the lipid-soluble polymer, the drug was immiscible and had a nonuniform distribution, resulting in crystallization on the surface of the fiber, which was found to be the main reason for burst release. The drug catalpol released easily in PBS because of its hydrophilicity. Therefore, the same solubility of the drugs and polymers are essential to avoid burst release. The release of protein can be varied by altering the method of electrospinning, e.g., blending or coaxial.16 Here, the catalpol was loaded into nanofibers by suspending them within a polymer solution. Catalpol is a hydrophilic drug, which is highly soluble in water and methanol, but sparingly soluble in lipophilic solvents. In our study, catalpol was dissolved in HFP firstly, and then mixed with PLGA. SEM images showed no crystal drug particle on the surface of fiber, which may be due to addition of silk fibroin. Fibroin protein is a
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water-soluble biological protein, which may enhance the solubility of catalpol in the composite polymers and promote catalpol release in 1 h. In addition, the degradation of the polymer also affects the rate of drug
release.25 In the present study, PLGA degradation caused a slightly acidic environment, which accelerated the release of catalpol. It was hard to detect catalpol with releasing at day 7, since catalpol was destructive
FIGURE 5. Immunocytochemistry analysis. hASCs were seeded on various catalpol-loaded scaffolds and cultured in H-DMEM. On day 3 (a) and day 5 (b), the cells had been detected expressing bIII-tubulin (green) and Nissl (red). Cell nuclei (blue) were counterstained with Hoechst 33258. Images were obtained by a laser scanning confocal microscope. TCP: tissue culture plate. Images were taken at 340 magnification. Scale bar indicates 100 lm and applies to all images.
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to acidic microenvironment induced by accumulation of acidic monomers and oligomers during PLGA degradation.14 Functionalized MWCNTs by grafting may help drug noncovalent interaction between nanotubes and the catalpol to make catalpol was easier to be absorbed by MWCNTs.29 Neuronal Differentiation of hASCs Promoted by Catalpol Cells were cultured for 5 days without any neurogenic inducer, and then immunofluorescence staining was performed to characterize the expression of bIIItubulin (neuronal marker) and Nissl body (a large granular body found in neurons). We observed a higher percentage of cells (approx. >60%) with neuronal phenotype on catalpol-loaded nanofibers expressing bIII-tubulin and Nissl, compared with control group at day 3 (Fig. 5a), and day 5 (Fig. 5b), respectively. These cells exhibited a stretched network of dendrites and elongated along a preferred axis, showing morphology indicative of neurons or neuronal progenitors. Introduction of catalpol enhanced the expression of bIII-tubulin and Nissl (Fig. 6), indicating catalpol’s neurogenic potential for hASCs. In particular, the expression of bIII-tubulin increased with culture time (Fig. 6a), but no significant differences were found; in contrast, the expression of Nissl significantly decreased, except for the control group (Fig. 6b). Additionally, with increase in the concentration of catalpol, the expression of bIII-tubulin and Nissl increased. However, cells with neuronal morphology were also presented but at dramatically lower fractions with catalpol of 5 mg/mL, indicating cytotoxicity of catalpol at higher concentration and longer culture time (Fig. 6b). Therefore, in the present experimental conditions, hASCs cultured on nanofibrous scaffolds with 4 mg/mL catalpol expressed significant higher b-tubulin III and Nissl than other groups. Interestingly, hASCs seeded on nanofibrous scaffolds without catalpol introduction remained undifferentiated with fibroblastic phenotype, but partially expressed bIII-tubulin and Nissl. Our results were somehow consistent with a previous report by Jang et al., in which they demonstrated that undifferentiated ASCs expressed low levels of MAP-2 (mature neural marker) and glial fibrillary acidic protein (GFAP, astrocyte marker), suggesting that ASCs may retain a native potential for neuronal differentiation.13 The expression of bIII-tubulin, in the cells that were cultured on the PLGA/MWCNTs/SF/catalpol scaffolds was evaluated by RT-PCR (Fig. 7). The expression of the bIII-tubulin gene was normalized to the expression of GADPH on the same samples. The expression of bIII-tubulin on PLGA/MWCNTs/SF with
FIGURE 6. Average percentage of positive hASCs with neuronal markers bIII-tubulin (a) and Nissl body (b) after cultured on various catalpol-loaded substrates. Positive cell numbers in each of ten images were counted for each sample. TCP: tissue culture plate. Data are represented as mean 6 SD. * and *** represents p < 0.05 and p < 0.001, compared with control group, respectively. #, ##, and ### represents p < 0.05, p < 0.01, and p < 0.001, compared with 4 mg/mL catalpol group, respectively.
4 mg/mL catalpol was significantly higher compared with all other groups. Interestingly, the expression level of bIII-tubulin was enhanced even under the nonneurogenic conditions by only culturing the cells on the nanofibrous scaffolds. This might be caused by the MWCNTs in the scaffold, since it has been reported that MWCNT sheet has a potential to be used for the purpose of neural regeneration by implantation of neural cells cultured on this sheet for the enhancement of cell function and guidance of cell migration.17 The results of immunoblotting assay showed that, the level of neuronal marker, bIII-tubulin, was upregulated on PLGA/MWCNTs/SF with 4 mg/mL catalpol compared with all other groups (Fig. 8). At present, the most common method to induce ASCs neural differentiation is based on two-dimensional culture supplemented with series of induction factors. For instance, ASCs could differentiate into neuronal-like cells by culturing them in the presence of
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FIGURE 7. RT-PCR analysis of bIII-tubulin expression by hASCs after seeding on PLGA/MWCNTs/SF nanofibrous composite scaffolds loaded with different concentrations of catalpol at day 3 of culture. The results showed a significant gene upregulation of bIII-tubulin on composite scaffolds with 4 mg/mL catalpol compared to all other groups. TCP: tissue culture plate. **, and *** represents p < 0.01, and p < 0.001, compared with TCP group, respectively. # and ### represents p < 0.05 and p < 0.001, compared with control group, respectively.
variety of therapeutic agents, such as drugs, antibiotics, growth factors, and proteins have been encapsulated in electrospun nanofibers physically and chemically, achieving controlled release behavior in vitro and in vivo.30 It is reasonable to encapsulate neural inductive reagents in electrospun nanofibers and continually release them to induce neural differentiation. The enhanced enhanced expression of neuronal specific markers has been demonstrated on retinoic acid (RA)-encapsulated electrospun fibers,15 or by using human glial cell-derived neurotrophic factor (GDNF)-encapsulated aligned electrospun fibrers in a rat model.6 Catalpol is an extract from the Chinese herb, and it has the neuroprotective effects. Besides attenuating lipopolysaccharide (LPS)-induced dopaminerrgic neurotoxicity,32 protecting mesencephalic neurons from MPP+ (1-methyl-4-phenylpyridinium)-induced oxidative stress,33 and increasing presynaptic proteins and upregulating signaling molecules levels in the aged rats,22 its potential in neurogenesis has been confirmed by our study, which make us to choose it as a neurogenesis inducer. In our study, catalpol-loaded PLGA/MWCNTs/SF composite nanofibers were fabricated for the first time by electrospinning process. However, the mechanism of catalpol for inducing stem cell into neuronal-like cells still remains unclear, and needs further study. Further research will also be focused on in vivo applications of electrospun scaffolds with catalpol, which would provide therapeutic benefits in nerve regenerative medicine. CONCLUSIONS
FIGURE 8. Immunoblot analysis of bIII-tubulin protein during hASCs neurogenesis on catalpol-loaded PLGA/MWCNTs/ SF matrix for 3 days. The band intensity was normalized to GAPDH. TCP: tissue culture plate. **, and *** represents p < 0.01, and p < 0.001, compared with TCP group, respectively. # and ### represents p < 0.05 and p < 0.001, compared with control group, respectively.
chemical induction reagents butylated hydroxyanisole (BHA) and dimethylsulfoxide (DMSO),35 or the cocktails of cytokines.37 Other protocols, such as neurospheres generation method,2 co-culture with Schwann cells34 have also been developed to induce ASCs into neuronal-like cells. On the other hand, besides the application as matrices, electrospun nanofibers also show promising future in drug release.27 A
In conclusion, PLGA/MWCNTs/SF nanofibers with various content of catalpol, a natural active ingredient, were successfully fabricated through electrospinning. By addition of catalpol, diameters of the nanofibers decreased and porosity increased. Moreover, the mechanical properties of the composite scaffolds were promoted. In particular, immunocytochemistry, RTPCR, and western blot analysis demonstrated more neuronal-like cells on scaffolds with 4 mg/mL catalpol. Our study confirmed for the first time that catalpol may possess great potential for neurogenesis and neural tissue engineering in future biomedical applications.
ACKNOWLEDGMENTS This work was funded by the Chinese National Natural Science Foundation (Nos. 81071009, 31200740, 81271412, 8141308), International S&T Cooperation Project of the Ministry of S&T of China
Neurogenesis of hASCs on Catalpol-Loaded Nanofibers
(No. 2010DFR30850), The People’s Livelihood S&T Project, Bureau of S&T of Dalian (Nos. 2010E11SF008, 2011E12SF030), and the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry.
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Potential Neurogenesis of Human Adipose-Derived Stem Cells on Electrospun Catalpol-Loaded Composite Nanofibrous Scaffolds JIAN-HUI GUO,1,2 YANG LIU,1,2 ZHENG-JUN LV,3 WEN-JUAN WEI,1 XIN GUAN,1 QING-LIN GUAN,4 ZHI-QIAN LENG,1 JING-YUAN ZHAO,1 HUI MIAO,1 and JING LIU1,2 1 Regenerative Medicine Center, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, People’s Republic of China; 2Institute of Integrative Medicine, Dalian Medical University, Dalian 116044, People’s Republic of China; 3Queen Mary University of London, London E1 4NS, UK; and 4Center Laboratory, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, People’s Republic of China
(Received 18 October 2014; accepted 23 March 2015) Associate Editor Peter E. McHugh oversaw the review of this article.
Abstract—Catalpol, a natural active ingredient extracted from the traditional Chinese medicine, was verified exhibiting beneficial effects on neural differentiation compared with commonly used chemical inducers by our previous studies. The aim of this study was to evaluate the effects of catalpolloaded scaffold on guiding neuronal differentiation of human adipose tissue-derived stem cells (hASCs). Fabrication technique of catalpol loading into the electrospun poly(lactic-coglycolic acid)/multi-walled carbon nanotubes/silk fibroin nanofibrous scaffolds was successfully established. The topographical and mechanical properties of the nanofibers scaffolds were characterized by scanning electron microscopy and tensile instrument, respectively. In vitro catalpol release was studied in phosphate-buffered solution at 37 C. Immunnocytochemistry, RT-PCR, and western blot assays were performed to estimate hASCs neuronal differentiation, and it was shown that catalpol has significantly upregulated the expressions of bIII-tubulin and Nissl. Our experiments demonstrated that catalpol, as a traditional Chinese medicine extract, could be encapsulated into composite nanofibers and induce differentiation of hASCs into neural-like cells, which might offer new avenues in nerve regeneration. Keywords—Electrospinning, Nanofibrous, Catalpol, Neurogenesis, Adipose tissue-derived stem cells.
INTRODUCTION Nerve injuries often result in severe damage since neural tissue possesses very limited capacity to regenerate new functional neurons. Tissue engineered neural Address correspondence to Jing Liu, Institute of Integrative Medicine, Dalian Medical University, Dalian 116044, People’s Republic of China. Electronic mail: [email protected] Jian-Hui Guo and Yang Liu have contributed equally to this work.
tissues may serve as a promising alternative for neural regeneration. Currently, clinical studies are focused on the delivery of stem cells capable of differentiation into neurons.5,8,9 However, such stem cells would need to proliferate and differentiate into the desired phenotype with the aid of adequate chemical, mechanical, or biological stimuli.28 Certain biomolecules, such as growth factors, genes, or drugs, can be incorporated within tissue engineering scaffolds to promote the proliferation and neuronal differentiation of the seeded cells, thereby enhancing nerve tissue regeneration.14 The electrospinning technique offers a possibility of incorporating protein growth factors or drugs within polymer nanofibers, which could then serve as a source of continued and controlled release of the growth factor or drugs.28 Electrospinning, a simple and versatile technique for preparing nanofibrous nonwoven mats, has been successfully developed to mimic the architecture of endogenous extracellular matrix (ECM). Various studies have confirmed that electrospun nanofibrous scaffolds could significantly facilitate cell adhesion and proliferation, drug loading, nutrients delivery, and waste removal due to their high porosity, spatial interconnectivity, and surface/volume ratio.12,23,30 The nanofibrous structures could also induce the extension of neurons, which are beneficial for during nerve regeneration.19,20 The electrospun nanofibers based on a wide range of biodegradable and biocompatible materials including natural proteins and synthetic polymers has been successfully prepared for tissue engineering applications. Among various synthetic polymers, poly(lactic-co-glycolic acid) (PLGA), approved by the US Food and Drug Administration for 2015 Biomedical Engineering Society
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several biomedical applications, has been widely used in tissue engineering.18 Silk fibroin (SF), one of strongest and toughest biological material, has become a promising biomaterial for tissue engineering and drug carrier due to its biocompatibility and the ability to control the release of growth factors.24 Carbon nanotubes (CNTs) are a conductive biomaterial with sizes comparable to ECM molecules such as collagens and laminins, which have been reported to favor neuronal growth.3 Both PLGA and SF could be fabricated into nanofibrous structures via electrospinning process, providing improved microenvironment for cell adhesion and proliferation. However, further improvement is still required when silk and PLGA are used in nerve tissue regeneration, where blending with conductive multi-walled carbon nanotubes (MWCNTs) in electrospun nanofibers might be an effective way. Adipose tissue is emerging as a source of stem cells obtained by less invasive methods such as lipoaspiration, and in larger quantities than bone marrow. Adipose tissue-derived stem cells (ASCs) have been found to have the potential in differentiating into functional neuronal-like cells.13 All of these advantages have made ASCs to be ideal cells for the treatment of neurological diseases. Catalpol, an iridoid glycoside isolated from the roots of traditional Chinese medical herb rehmannia, has been widely used as a traditional Chinese medicine for the treatment of stroke and diseases of aging owing to its neuroprotective effects. It has been reported that catalpol can induce neuronal differentiation in PC12 cells through activation of the intracellular signal transduction pathway, and promote neurite length.36 Our previous studies further confirmed that catalpol could improve neuronal differentiation and cell viability of human ASCs in vitro under two-dimensional condition, indicating promising future of catalpol as a neurogenesis inducer (unpublished data). Therefore, incorporation of catalpol into a composite nanofibrous scaffolds may improve their clinical efficacy in nerve tissue engineering. In our present study, catalpol was loaded into PLGA/MWCNTs/SF electrospun composite to directly induce hASCs into neuronal-like cells. The morphology, diameter distribution, and porosity of the fibers were studied. In addition, the mechanical properties of the composite nanofibrous scaffolds were characterized at dry and wet state, respectively. The release behaviors of catalpol and its influence on neuronal differentiation of hASCs were studied to assess the feasibility of the composite as neural regeneration matrice. To our knowledge, this is the first report as regard to the neurogenesis effects study of the electrospun catalpol-loaded composite nanofiber scaffolds.
MATERIALS AND METHODS Chemicals PLGA (85:15, Mw = 100,000) was obtained from Daigang Biomaterials Company (Jinan, China). 1,1,1,3,3,3-Hexafluor-2-propanol (HFP) was purchased from Xiya Reagent Company (Chengdu, China). Silk fibroin (SF) was obtained from Chinatiansi Biotek Company (Huzhou, China). MWCNTs were purchased from Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences (Chengdu, China); Catalpol (purity > 99%) was obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Fabrication of PLGA/MWCNTs/SF/Catalpol Nanofibers PLGA/MWCNTs/SF/catalpol composite nanofibers were fabricated using the electrospinning technique. The composite PLGA/MWCNTs/SF suspensions with catalpol at various concentrations were obtained by mixing the PLGA, MWCNTs, SF and catalpol solutions together (3:1:2:1, volume ratio). Firstly, PLGA was dissolved in HFP and incubated in 37 C water bath overnight. SF was added into HFP and stirred continuously for 60 min at room temperature. Meanwhile, MWCNTs and catalpol were dissolved in HFP under ultra-sonication for 60 and 10 min, respectively. Finally, the suspensions were stirred continuously for 30 min before electrospinning. Herein, the concentrations of PLGA, MWCNTs, and SF in the electrospinning solutions were fixed at 15, 0.2 and 10% (w/v), respectively. And the concentrations of catalpol were 0, 1, 2, 3, 4, and 5 mg/mL, respectively. The scaffolds without catalpol were set as the control group. The polymer solutions were then fed into a 10 mL syringe (BD, China) coupled with to a 9 G stainless steel needle using a syringe pump (LongerPump, China) at a flow rate of 1.0 mL/h. High voltage (Teslaman, China) of 15 kV was applied, and the fibers were collected on an aluminum foil wrapped collector kept at a distance of 10 cm from the needle tip. Collected nanofibers were dried overnight and used for the characterization and cell culture experiments. Morphology and Characterization of Electrospun Nanofibers Scanning Electron Microscopy The morphology of electrospun nanofibers was observed under scanning electron microscope (SEM) (JEOL-JSM 6360-LV, Japan) at an accelerating voltage
Neurogenesis of hASCs on Catalpol-Loaded Nanofibers
of 20 kV, after coating with a thin gold layer. For local high magnification, another SEM (SUPRA 55, Zeiss, Germany) was also used. The average diameter of the fibers was analyzed by an image analysis software (Image J, National Institute of Health, USA) based on the SEM images. At least 200 nanofibers from different images were determined for each sample. Surface Porosity Determination The surface porosity was calculated based on SEM images.1 Briefly, the porosity was determined by calculating volume beneath the surface and volume beneath the same surface. SEM images were firstly converted into two-dimensional matrix in gray scale format and it was assumed that the height was equal to maximum height of any point on the sample’s surface. Then the surface porosity calculation was processed by OriginPro software (OriginLab Corp, USA). Mechanical Property Study Tensile properties of electrospun nanofibers in the dry and wet state were determined using a tensile tester (Instron 5989, Canton, USA) at room temperature. The specimens were cut into rectangular pieces with the dimensions of 50 mm 9 10 mm, and the gage length of 10 mm. The thickness of each specimen was measured with a micrometer before testing and the distance between the gripping points was 30 mm. The mechanical testing was conducted with the grips moving at a stretch speed of 20 mm/min. A minimum of five strips from different sites of each sample were tested, and Young’s modulus, yield strength, and elongation at break were calculated. For the measurement in the wet state, the samples were soaked in phosphate-buffered solution (PBS) at 37 C for 1 h and then measured as described above. In Vitro Release of Catalpol from Nanofibers The electrospun samples were cut into square pieces of 25 mm 9 25 mm, and their weights were accurately measured. Then the samples were placed in centrifuge tubes containing 10 mL phosphate-buffered saline (PBS, pH 7.4), and were incubated in a 37 C water bath with constant sharking rate at 60 cycles/min for a period of 7 days. 1 mL solution was taken out from three tubes (triplicates) and 1 mL fresh PBS was added back at scheduled time points. The collected solutions were stored at 220 C, and thawed at room temperature before analysis. After filtration through a 0.22 lm membrane (Millipore, USA), samples were analyzed using high performance liquid chromatography (HPLC) to determine the amount of catalpol released as a function of time. The column type was
Hypersil C18 (5 lm of 200 mm 9 4.6 mm internal diameter). The solvent for calibration was 0.5 mg/mL catalpol in the solvent of acetonitrile and water (0.5:99.5, v/v), k = 210 nm. The percentage of the detectable catalpol release was determined using a preestablished standard curve based on the initial weight of catalpol incorporated in the nanofibers. Standard curve was constructed in the range 1.0–500.0 lg/mL for catalpol. The regression equation was calculated and the coefficient was determined (r2 = 0.9997). Human ASCs Culture and Seeding on Scaffolds Human ASCs were isolated and cultured as mentioned in the previous method.10 Briefly, adipose tissue was obtained from human liposuction surgery with informed consent, under the requirements of Ethic Committee of the First Affiliated Hospital of Dalian Medical University. The adipose tissues were washed extensively with PBS and then minced into 1 mm3 pieces. The adipose tissues were digested with collagenase I at 37 C for 60 min, and neutralized by adding human Adipose-derived Stem Cell Growth Medium (OriCellTM, Cyagen Biosciences, China). The digested adipose tissues were filtered with a nylon membrane (Fisher, USA) to remove undigested fragments and then the cellular suspension was centrifuged at 1000 rpm for 10 min. After three washes with PBS (centrifuged at 1000 rpm, 5 min), cells were incubated at 37 C in a humidified atmosphere containing 5% CO2, and medium was changed every 3 days. hASCs of passage 4–6 were used in the following experiments. Before cell seeding, the electrospun PLGA/ MWCNTs/SF/catalpol scaffolds were sterilized under ultraviolet light for 30 min on each side, and washed twice with PBS for 5 min each to remove residual solvent. Then the fibers were subsequently immersed in High-glucose Dulbecco’s Modified Eagle’s Medium (H-DMEM) (Invitrogen, USA) overnight before cell seeding. hASCs grown in 25 cm2 cell culture flasks (Corning, USA) were detached by 0.25% trypsin– 0.1% ethylene diaminotetraacetic acid (EDTA) (Invitrogen, USA). A density of 5000 cells/well were seeded on the scaffold in a 24-well plate (Corning, USA) and cultured in H-DMEM supplemented with 1% penicillin–streptomycin (Invitrogen, USA; termed as ‘‘hASC growth media’’). Cells seeded on tissue culture polystyrene (TCP) in 24-well plates cultured in Adipose-derived Stem Cell Growth Medium at the same density were served as negative control. Immunocytochemistry for hASCs Neurogenesis After 3 and 5 days culture on electrospun PLGA/ MWCNTs/SF/catalpol nanofibers in ‘‘hASC growth
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media’’, immunocytochemistry for hASCs neurogenesis was performed. The cells were initially fixed with 4% paraformaldehyde for 30 min at room temperature, and then permeabilized with 0.1% Triton X100 for 10 min and washed three times with PBS. Nonspecific antibody binding was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and then the cells were exposed to rabbit antiIII-Tubulin (1:1000) overnight at 4 C. After washed for three times with PBS, the cells were incubated with goat anti-rabbit fluorescein isothiocyanate secondary antibody (FITC, 1:1000) (all from Sigma, USA) for 1 h at room temperature, and then washed with PBS again. Finally, the samples were stained with Nissl-PE (1:200, Molecular, USA) for 15 min, and the nuclei were then stained with Hoechst 33258 (Sigma, USA) for 15 min, followed by three washes with PBS. Fluorescence images were taken by a laser scanning confocal microscope (Leica SP8, Germany). At least ten images were chosen randomly from each staining, and the number of positive cells were calculated and analyzed.
Real-Time Polymerase Chain Reaction (RT-PCR) Analysis RNA was isolated using the RNAiso Plus (Takara, Japan). RNA concentration was measured using the NanoDrop 2000 (Thermo, USA). Total RNA (1 mg) was subjected to reverse transcription (RT) by PrimeScript RT reagent Kit (37 C for 15 min, 85 C for 5 s) (Takara, Japan). Primers were designed by PRIMER EXPRESS software (PE Applied Biosystems, USA). Quantitative real-time PCR was performed using Light-Cycler 480 (Roche Applied Science, USA) with SYBR Green I (Takara, Japan) as the detection system. After denaturing for 30 s at 95 C, amplification was performed with 40 cycles of 5 s at 95 C and 20 s at 56 C. Melting curve analysis was performed with 1 s at 95 C, 15 s at 65 C, and 1 s at 95 C, melting curve analysis was used to confirm the specificity of amplification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a standard control for the normalization of the gene expression level, and the gene expression levels of the cells on the TCP were used as a negative control group. Results were analyzed by the value of DDCT using Light-Cycler software (Roche, USA). The sequences of PCR primers (forward and backward, 5¢–3¢) were as follows: bIII-tubulin: forward 5¢-GAACCCGGAACCAT GGACAG-3¢ reverse 5¢-GACCCTTGGCCCAGT TGTTG-3¢
GAPDH: forward 5¢-AACGGATTTGGTCGTA TTGGG-3¢ reverse 5¢-TGGAAGATGGTGATGG GATTT-3¢
Western Blot Analysis hASCs were removed from nanofibrous composite scaffolds, and lysed with RIPA buffer. Protein concentration was measured using BCA protein kit. Extracts were re-suspended with 59 SDS-polyacrylamide (PAGE) gel buffer (all from Beyotime Biotech, China) and boiled for 7–8 min. Equal amounts of protein extracts (20–30 mg) were fractioned on a 10% SDSPAGE. Following electrophoresis, the proteins were transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, USA). After the membrane was blocked with blocking buffer (0.1% Tween 20, and 5% skim milk in PBS, pH 7.4) for 1 h, the membrane was incubated overnight at 4 C with anti-bIII-tubulin (Sigma, USA) diluted 1:1500 in TBST (Tris-buffered saline-Tween 20) and anti-GAPDH primary antibodies at 4 C for overnight. After washing three times with TBST, the membrane was incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (diluted 1:1000 in TBST, Abcam, USA). After washing the membrane, labeled proteins were visualized with enhanced chemiluminescence (ECL) detection reagents (Amersham Pharmacia Biotech., USA) and were analyzed using an imaging analysis system (Gelpro32, MediaCybernetics, USA). Statistical Analysis Experiments were performed with triplicate samples and all data were presented as mean ± standard deviation (SD). Single factor analysis of variance (ANOVA) was performed to determine statistical significance and Fisher’s LSD for multiple comparisons. The statistical software package SPSS 13.0 (SPSS, Chicago, IL, USA) was used for the data analysis. A value of p < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION Characterization of the Composite Electrospun Nanofibrous Scaffolds PLGA/MWCNTs/SF/catalpol nanofibers fabricated by electrospinning showed nonwoven mat structures and random fiber distribution with smooth and beadless fibers morphology, as shown in Fig. 1. Higher magnification SEM images (A3–F3, Fig. 1) revealed
Neurogenesis of hASCs on Catalpol-Loaded Nanofibers
that the nanofibers were with smooth surface and no catalpol particles on the fiber surfaces, indicating that catalpol distributed uniformly in nanofibers and verifying PLGA/MWCNTs/SF/catalpol combination is a reliable and reproducible electrospinning system. The diameter distribution of the fibers (Fig. 1A4–F4) showed diameter of nanofibers decreased nonlinearly with the increase of catalpol concentration. The diameters of the electrospun nanofibers have an influence
on cell behavior, which are dependent on the electrospinning conditions, such as concentration of the polymer solutions, voltage, flow rates and the collecting distance. Christopherson et al. have investigated the influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation and demonstrated that as the fiber diameter decreased, higher degree of proliferation and cell spreading were observed.7 In this study, incorporation of catalopl
FIGURE 1. Morphology characterization of composite electrospun nanofibers by SEM. Nanofibrous scaffolds were electrospun at distance of 10 cm and flow rate of 1.0 mL/h. Catalpol concentration was 0, 1, 2, 3, 4, and 5 mg/mL, respectively. PLGA/MWCNTs/ SF catalpol-loaded scaffolds exhibited fairly uniform fibers without the presence of beads along the fibers. A1–F1: low magnification (31000); A2–F2: 310,000; A3–F3: high magnification (3200,000); A4–F4 showed the diameter distribution histogram of the composite nanofibers.
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FIGURE 2. The porosity of nanofibers analyzed by Image J software from SEM iamges. **, and *** represents p < 0.01, and p < 0.001, compared with control group, respectively.
decreased the diameter of nanofibers. The explanation may be that addition of small molecules, such as catalpol, could increase the concentration of charged particles, thus raise the electric force of the polymer solution, and facilitate decreased fiber diameter. Zeng et al. reported that the diameters of the fibers were sharply decreased, since the addition of drugs (such as rifampin) had reduced the surface tension,38 which was consistent with our results. A perfect three-dimensional nanofibrous scaffold can afford not only cell attachment, proliferation, and differentiation, but also sufficient transport for nutrients and waste removal.21 Nanofibrous scaffolds with high porosity can promote cells growth and provide a three-dimensional structure similar to native tissue. The porosity of PLGA/MWCNTs/SF nanofibers with different concentrations of catalpol was calculated according to the method demonstrated by Abdullah and Khairurrijal.1 As demonstrated in Fig. 2, the porosities of PLGA/MWCNTs/SF nanofibers with 3 mg/mL and 4 mg/mL of catalpol were significantly higher than those in control group. The porosities of nanofibers were 60.35 ± 0.23, 61.61 ± 1.46, and 50.67 ± 1.83%, respectively, for nanofibers with 3 mg/ mL, 4 mg/mL catalpol, and the control group. However, there was no significant difference in nanofiber porosities between control group and the other experimental groups. Our results showed that catalpol has maintained the high porosity of the composite scaffolds. The values of Young’s modulus, yield strength, and elongation at break of the samples were determined and illustrated in Fig. 3. We expect that electrospun PLGA/MWCNTs/SF composite nanofibers could display excellent mechanical properties. Results of mechanical evaluations showed that the Young’s modulus of nanofibrous scaffolds were greatly im-
FIGURE 3. Mechanical properties of electrospun composite scaffolds with different concentrations of catalpol at dry and wet state. (a) Young’s modulus; (b) yield strength; (c) elongation at break. *, **, and *** represents p < 0.05, p < 0.01, and p < 0.001, compared with control group, respectively. All values are represented as mean 6 SD (n 5 5).
proved with the introduction of catalpol, thus creating a biocomposite scaffold with superior tensile properties, which was especially suitable for nerve tissue engineering (Fig. 3a). There were no significant differences among groups with regards to yield strength when the scaffolds were in dry state (Fig. 3b). The elongation at break of nanofibers containing catalpol
Neurogenesis of hASCs on Catalpol-Loaded Nanofibers
was significantly higher than that of control group, except for the 1 mg/mL catalpol group (Fig. 3c). With the introduction of catalpol, the hydrophilicity of PLGA/MWCNTs/SF membrane was increased, causing the decrease of Young’s modulus in wet state. After incubation in PBS, the electrospun composite membrane exhibited a significant increase in both of yield strength and elongation at break with respect to those in dry state. Mechanical properties of nanofibrous scaffolds are essential for tissue engineering. In particular, appropriate mechanical properties can help with providing support and guidance for axons regeneration. The ideal scaffolds are ones with mechanical properties close to in vivo tissues, and this is the reason we chose MWCNTs as a composition in the polymer solution, aiming to enhance the overall mechanical properties of the scaffolds.
FIGURE 4. In vitro release profile of catalpol from electrospun composite scaffolds with different concentrations of catalpol. The scaffolds were incubated in PBS (pH 7.4) at 37 °C, and the samples were analyzed with HPLC to determine the amount of catalpol released as a function of time. ** represents p < 0.01 compared with the other groups.
In Vitro Catalpol Release Incorporation of growth factors,16 vitamins,30 or antibiotics into biocompatible and biodegradable nanofibers might be a promising alternative to regulate cell proliferation and differentiation. Catalpol has exhibited various biological activities, including antioxidation, anti-apoptosis, and radio-protection,4 and was loaded in electrospun nanofibers to regulate cell behaviors in our study. The controlled release of catalpol from the composite nanofibrous scaffolds was analyzed by HPLC, and their release profiles from electrospun nanofibers at a temperature of 37 C and pH 7.4 were shown in Fig. 4. The release profile of catalpol had three stages: an initial burst release, further a constant stage, followed by the decrease. The catalpol released quite fast during the initial 1 h, and then maintained a slight decreasing stage from 1 to 24 h. After 24 h, the release rate slowed down until no catalpol could be detected at day 7. As shown in Fig. 4, the highest detectable drug release was over 51.20 ± 5.65%, which appeared in the nanofibrous scaffolds loaded with 5 mg/mL. The initial release percentages of nanofibers with different concentrations of catalpol at 1 h were around 50%, the lowest value was 48.61 ± 5.36% of nanofibers with 3 mg/mL catalpol. From 1 to 12 h, release percentages decreased in all groups, and on the contrary, during 12–24 h, the release rate remained stable in each group. The detectable catalpol release of electrospun nanofibers with 1 mg/mL catalpol was significantly lower than that of the other groups at 4, 12, and 24 h. From 24 h to 5 days, all groups sustained downward trends to the lowest point, with the lowest value of 9.38 ± 2.88 and 19.18 ± 4.41% for 1 and 5 mg/mL catalpol group, respectively. It is obvious that the ini-
tial releases were all above 50%, which is the common problem caused by blending, namely burst release. The initial burst release results in hard control of sustained drug release, therefore it is necessary to avoid burst release;15 however, it may be useful for drug release system which requires a large initial dose and long-term maintenance of the drug. Researcher are making efforts to avoid burst release using the methods of shell–core electrospinning,15 mixing the drug with the polymer by chemical binding,26 multilayered continuous electrospinning.11 Zeng et al. have explained that burst release phenomenon occurred due to the differences in dissolution characteristics of the drug and its associated polymer.38 In their work, paclitaxel, doxorubicin base were lipid-soluble drugs, while doxorubicin hydrochloride was water-soluble drug. When the water-soluble drug was blended with the lipid-soluble polymer, the drug was immiscible and had a nonuniform distribution, resulting in crystallization on the surface of the fiber, which was found to be the main reason for burst release. The drug catalpol released easily in PBS because of its hydrophilicity. Therefore, the same solubility of the drugs and polymers are essential to avoid burst release. The release of protein can be varied by altering the method of electrospinning, e.g., blending or coaxial.16 Here, the catalpol was loaded into nanofibers by suspending them within a polymer solution. Catalpol is a hydrophilic drug, which is highly soluble in water and methanol, but sparingly soluble in lipophilic solvents. In our study, catalpol was dissolved in HFP firstly, and then mixed with PLGA. SEM images showed no crystal drug particle on the surface of fiber, which may be due to addition of silk fibroin. Fibroin protein is a
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water-soluble biological protein, which may enhance the solubility of catalpol in the composite polymers and promote catalpol release in 1 h. In addition, the degradation of the polymer also affects the rate of drug
release.25 In the present study, PLGA degradation caused a slightly acidic environment, which accelerated the release of catalpol. It was hard to detect catalpol with releasing at day 7, since catalpol was destructive
FIGURE 5. Immunocytochemistry analysis. hASCs were seeded on various catalpol-loaded scaffolds and cultured in H-DMEM. On day 3 (a) and day 5 (b), the cells had been detected expressing bIII-tubulin (green) and Nissl (red). Cell nuclei (blue) were counterstained with Hoechst 33258. Images were obtained by a laser scanning confocal microscope. TCP: tissue culture plate. Images were taken at 340 magnification. Scale bar indicates 100 lm and applies to all images.
Neurogenesis of hASCs on Catalpol-Loaded Nanofibers
to acidic microenvironment induced by accumulation of acidic monomers and oligomers during PLGA degradation.14 Functionalized MWCNTs by grafting may help drug noncovalent interaction between nanotubes and the catalpol to make catalpol was easier to be absorbed by MWCNTs.29 Neuronal Differentiation of hASCs Promoted by Catalpol Cells were cultured for 5 days without any neurogenic inducer, and then immunofluorescence staining was performed to characterize the expression of bIIItubulin (neuronal marker) and Nissl body (a large granular body found in neurons). We observed a higher percentage of cells (approx. >60%) with neuronal phenotype on catalpol-loaded nanofibers expressing bIII-tubulin and Nissl, compared with control group at day 3 (Fig. 5a), and day 5 (Fig. 5b), respectively. These cells exhibited a stretched network of dendrites and elongated along a preferred axis, showing morphology indicative of neurons or neuronal progenitors. Introduction of catalpol enhanced the expression of bIII-tubulin and Nissl (Fig. 6), indicating catalpol’s neurogenic potential for hASCs. In particular, the expression of bIII-tubulin increased with culture time (Fig. 6a), but no significant differences were found; in contrast, the expression of Nissl significantly decreased, except for the control group (Fig. 6b). Additionally, with increase in the concentration of catalpol, the expression of bIII-tubulin and Nissl increased. However, cells with neuronal morphology were also presented but at dramatically lower fractions with catalpol of 5 mg/mL, indicating cytotoxicity of catalpol at higher concentration and longer culture time (Fig. 6b). Therefore, in the present experimental conditions, hASCs cultured on nanofibrous scaffolds with 4 mg/mL catalpol expressed significant higher b-tubulin III and Nissl than other groups. Interestingly, hASCs seeded on nanofibrous scaffolds without catalpol introduction remained undifferentiated with fibroblastic phenotype, but partially expressed bIII-tubulin and Nissl. Our results were somehow consistent with a previous report by Jang et al., in which they demonstrated that undifferentiated ASCs expressed low levels of MAP-2 (mature neural marker) and glial fibrillary acidic protein (GFAP, astrocyte marker), suggesting that ASCs may retain a native potential for neuronal differentiation.13 The expression of bIII-tubulin, in the cells that were cultured on the PLGA/MWCNTs/SF/catalpol scaffolds was evaluated by RT-PCR (Fig. 7). The expression of the bIII-tubulin gene was normalized to the expression of GADPH on the same samples. The expression of bIII-tubulin on PLGA/MWCNTs/SF with
FIGURE 6. Average percentage of positive hASCs with neuronal markers bIII-tubulin (a) and Nissl body (b) after cultured on various catalpol-loaded substrates. Positive cell numbers in each of ten images were counted for each sample. TCP: tissue culture plate. Data are represented as mean 6 SD. * and *** represents p < 0.05 and p < 0.001, compared with control group, respectively. #, ##, and ### represents p < 0.05, p < 0.01, and p < 0.001, compared with 4 mg/mL catalpol group, respectively.
4 mg/mL catalpol was significantly higher compared with all other groups. Interestingly, the expression level of bIII-tubulin was enhanced even under the nonneurogenic conditions by only culturing the cells on the nanofibrous scaffolds. This might be caused by the MWCNTs in the scaffold, since it has been reported that MWCNT sheet has a potential to be used for the purpose of neural regeneration by implantation of neural cells cultured on this sheet for the enhancement of cell function and guidance of cell migration.17 The results of immunoblotting assay showed that, the level of neuronal marker, bIII-tubulin, was upregulated on PLGA/MWCNTs/SF with 4 mg/mL catalpol compared with all other groups (Fig. 8). At present, the most common method to induce ASCs neural differentiation is based on two-dimensional culture supplemented with series of induction factors. For instance, ASCs could differentiate into neuronal-like cells by culturing them in the presence of
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FIGURE 7. RT-PCR analysis of bIII-tubulin expression by hASCs after seeding on PLGA/MWCNTs/SF nanofibrous composite scaffolds loaded with different concentrations of catalpol at day 3 of culture. The results showed a significant gene upregulation of bIII-tubulin on composite scaffolds with 4 mg/mL catalpol compared to all other groups. TCP: tissue culture plate. **, and *** represents p < 0.01, and p < 0.001, compared with TCP group, respectively. # and ### represents p < 0.05 and p < 0.001, compared with control group, respectively.
variety of therapeutic agents, such as drugs, antibiotics, growth factors, and proteins have been encapsulated in electrospun nanofibers physically and chemically, achieving controlled release behavior in vitro and in vivo.30 It is reasonable to encapsulate neural inductive reagents in electrospun nanofibers and continually release them to induce neural differentiation. The enhanced enhanced expression of neuronal specific markers has been demonstrated on retinoic acid (RA)-encapsulated electrospun fibers,15 or by using human glial cell-derived neurotrophic factor (GDNF)-encapsulated aligned electrospun fibrers in a rat model.6 Catalpol is an extract from the Chinese herb, and it has the neuroprotective effects. Besides attenuating lipopolysaccharide (LPS)-induced dopaminerrgic neurotoxicity,32 protecting mesencephalic neurons from MPP+ (1-methyl-4-phenylpyridinium)-induced oxidative stress,33 and increasing presynaptic proteins and upregulating signaling molecules levels in the aged rats,22 its potential in neurogenesis has been confirmed by our study, which make us to choose it as a neurogenesis inducer. In our study, catalpol-loaded PLGA/MWCNTs/SF composite nanofibers were fabricated for the first time by electrospinning process. However, the mechanism of catalpol for inducing stem cell into neuronal-like cells still remains unclear, and needs further study. Further research will also be focused on in vivo applications of electrospun scaffolds with catalpol, which would provide therapeutic benefits in nerve regenerative medicine. CONCLUSIONS
FIGURE 8. Immunoblot analysis of bIII-tubulin protein during hASCs neurogenesis on catalpol-loaded PLGA/MWCNTs/ SF matrix for 3 days. The band intensity was normalized to GAPDH. TCP: tissue culture plate. **, and *** represents p < 0.01, and p < 0.001, compared with TCP group, respectively. # and ### represents p < 0.05 and p < 0.001, compared with control group, respectively.
chemical induction reagents butylated hydroxyanisole (BHA) and dimethylsulfoxide (DMSO),35 or the cocktails of cytokines.37 Other protocols, such as neurospheres generation method,2 co-culture with Schwann cells34 have also been developed to induce ASCs into neuronal-like cells. On the other hand, besides the application as matrices, electrospun nanofibers also show promising future in drug release.27 A
In conclusion, PLGA/MWCNTs/SF nanofibers with various content of catalpol, a natural active ingredient, were successfully fabricated through electrospinning. By addition of catalpol, diameters of the nanofibers decreased and porosity increased. Moreover, the mechanical properties of the composite scaffolds were promoted. In particular, immunocytochemistry, RTPCR, and western blot analysis demonstrated more neuronal-like cells on scaffolds with 4 mg/mL catalpol. Our study confirmed for the first time that catalpol may possess great potential for neurogenesis and neural tissue engineering in future biomedical applications.
ACKNOWLEDGMENTS This work was funded by the Chinese National Natural Science Foundation (Nos. 81071009, 31200740, 81271412, 8141308), International S&T Cooperation Project of the Ministry of S&T of China
Neurogenesis of hASCs on Catalpol-Loaded Nanofibers
(No. 2010DFR30850), The People’s Livelihood S&T Project, Bureau of S&T of Dalian (Nos. 2010E11SF008, 2011E12SF030), and the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry.
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