Synergistic Promotion of Blood Vessel Regeneration by Astragaloside

Apr 24, 2013 - E-mail: [email protected]. ... The promotion of blood vessel initiation and growth plays an important role in the realization of therap...
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Synergistic Promotion of Blood Vessel Regeneration by Astragaloside IV and Ferulic Acid from Electrospun Fibrous Mats Huan Wang,†,‡,§ Yun, Zhang,†,§ Tian Xia,∥ Wei Wei,∥ Fang Chen,† Xueqin Guo,† and Xiaohong Li*,†,‡ †

Key Laboratory of Advanced Technologies of Materials, Ministry of Education of China, School of Materials Science and Engineering, and ‡School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China ∥ Department of Pathology, The 452nd Hospital of People’s Liberation Army, Chengdu 610021, China ABSTRACT: The promotion of blood vessel initiation and growth plays an important role in the realization of therapeutic vascularization and regeneration of functional tissues. Astragalus membranaceus and angelica sinensis are commonly used traditional Chinese medicines for enriching the blood. In the current study astragaloside IV (AT, the main active ingredient of astragalus) and ferulic acid (FA, the main ingredient of angelica) were loaded into electrospun fibrous scaffolds to provide abundant and sustained biological factors required to initiate vascularization and bring it to maturity. The cell viability after AT and FA treatment was dose-dependent with an optimal concentration of around 50 μg/mL, and the most significant synergistic effect was demonstrated for the combined treatment with AT and FA with the ratio of 7/3 on both primary endothelial and smooth muscle cells. The in vitro release study showed that the amount of AT and FA release could be regulated by their loading amount and ratios in electrospun fibers. The localized and sustained codelivery of AT and FA indicated significantly high cell viability and secretion of extracellular matrices for both endothelial and smooth muscle cells, and induced significantly high densities of vascular structures after subcutaneous implantation. The most significant angiogenesis promotion with few inflammatory reactions was demonstrated for electrospun fibers containing AT and FA with the ratio of 7/3. It was suggested that the integration of the synergistic effect of Chinese medicine into electrospun fibrous scaffolds should provide clinical relevance for therapeutic vascularization, full vascularization in engineered tissues, and regeneration of blood vessel substitutes. KEYWORDS: electrospun fibers, traditional Chinese medicine, tissue engineering scaffold, blood vessel

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INTRODUCTION Blood vessels of the body are responsible for the input and exchange of nutrients and oxygen, the output of metabolism, and other important functions. Promoting the process of angiogenesis is very crucial for improving blood perfusion for the treatment of ischemic disease, such as coronary heart disease and myocardial infarction.1 In addition, the creation of adequate blood vessels remains an essential prerequisite for the establishment of tissue engineering constructs, since a tissue that is more than a few millimeters in size generally cannot survive by only the diffusion of nutrients and metabolic products.2 However, the lack of control over microvasculature formation remains a key roadblock to the realization of therapeutic vascularization and regeneration of functional tissues. There are two main strategies utilized to engineer vascularized tissues. One of them is the utilization of endothelial cells (ECs) and their ability to form new vessels, and the other is based on scaffold-based techniques, such as biologically derived vessels and synthetic tubular scaffold.3 It is worth noting that abundant angiogenic growth factors are usually required in the above attempts to activate ECs and smooth muscle cells (SMCs), and to initiate and sustain a lot of © 2013 American Chemical Society

cellular activities, including cell migration, proliferation, invasion, and formation of tubular structures.4 A lot of biological factors have been identified to promote vascularization, and most of them are protein growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), and plasmids DNA (pDNA) encoding these growth factors.5 The soluble biological factors typically have a short half-life, and direct delivery of growth factors or using gene therapy approaches has not yielded the expected results in clinical practice, which makes it necessary to stabilize them during their supply. Several strategies have been adopted to integrate these biological factors into tissue engineering scaffolds to achieve local and sustained delivery, and consequently a prolonged biological effect with induction of tissue revascularization. Formiga et al. indicated an increase in angiogenesis and arteriogenesis in a rat model of ischemia− reperfusion treated with poly(lactic-co-glycolic acid) microReceived: Revised: Accepted: Published: 2394

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scaffolds. Alignment and elongation of SMCs on random nonwoven nanofibrous silk scaffolds were observed within 5 days after seeding, and a complex interconnecting network of capillary tubes with identifiable lumens by day 7.18 Montero et al. loaded bFGF into electrospun gelatin scaffolds, indicating that EC proliferation and capillary formation were directly proportional to the bFGF loading concentrations from 0 to 100 ng/mL.19 He et al. constructed core−sheath electrospun fibers with the encapsulation of plasmids encoding FGF and VEGF. The sustained release of pDNA from fibrous mats promoted protein expression and enhanced ECM secretion and microvessel generation. But low cell viability, strong inflammation, and necrotic tissues were indicated for electrospun fibers with loaded pDNA polyplexes after subcutaneous implantation.20 This study was aimed to assess electrospun fibers with TCM loadings to allow a localized and sustained delivery for an efficient vascularization. The half-life of AT was 34−131 min in rats and 50−68 min in dogs, indicating that AT may be quickly eliminated.21 The establishment of a sustained release system is a booming field of TCM pharmaceutical research since it can improve the pharmacological effects and reduce the adverse reactions.22 On the other hand, TCM is characterized by the use of herbal formulas that are usually grouped by two or more medicinal herbs, which can effectively reduce side effects and produce synergetic effects to be greater than the sum of the individual effects.23 Therefore, in the current study AT and FA were simultaneously loaded into electrospun fibers, and the dose ratios of AT and FA were optimized to enhance cell adhesion, proliferation, and ECM secretion of primary ECs and SMCs. The synergistic effects of AT and FA on the extent of vascularization and vessel maturation were evaluated after subcutaneous implantation into Sprague−Dawley rats.

particles loaded with VEGF, but not in the empty microparticles or free-VEGF groups after one month treatment.6 However, the denaturation related to storage or scaffold formulation and the high price of purified growth factors are factors restricting the application to inductive tissue engineering scaffolds.7 Alternatively, pDNA encoding angiogenic growth factors were loaded into tissue engineering scaffolds, and polyplexes of pDNA and polycations were utilized to induce sufficient protein expression. Guo et al. prepared complexes of VEGF-encoded pDNA with N,N,N-trimethyl chitosan chloride, which were loaded into a bilayer porous collagen−chitosan/ silicone membrane as dermal equivalents for treatment of fullthickness burn wounds. The loading of pDNA complexes resulted in a significantly higher number of newly formed and mature blood vessels, and fastest regeneration of the dermis.8 However, it is still a major challenge to achieve a balance between the cytotoxicity and transfection efficiency of the geneactivated matrix.9 Attempts have been made to promote vascularization by other biological factors, and one of them is the utilization of traditional Chinese medicines (TCMs), which are gradually becoming accepted by more people worldwide for their effectiveness, weak side effects, and safety.10 There are a lot of related theories about angiogenesis, and many TCMs have been employed in the field of blood circulation promotion. For example, the Chinese medicine Huangqi has been demonstrated to nourish the “Blood” (the body circulation) and raise “Qi” (the vital energy) for the treatment of blood-borne diseases. The major active constituent is astragaloside IV (AT), a 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol, which has been reported to be used clinically for treatment of cardiovascular disorders and for the repair of injured organs and tissues.11 Zhang et al. indicated that AT could stimulate cell proliferation, significantly increase the mean tube length of ECs in Matrigel, and enhance mRNA expression of VEGF and VEGF receptors.12 Chick chorioallantoic membrane assay showed an increase in the vessel tube formation, and in the expression and accumulation of hypoxia-inducible factor-1 and VEGF after AT treatment.13 Other Chinese herbal medicines, such as ligusticum chuanxiong hort and angelica sinensis, are clinically used to treat angina pectoris and hypertensive diseases through pain relief and promoting blood circulation. Ferulic acid (FA), a 4-hydroxy-3-methoxycinnamic acid, is the main efficacious ingredient, indicating significant effects to improve blood fluidity, inhibit platelet aggregation, and exhibit strong antioxidant activity.14 Wang et al. found that FA could significantly improve cell proliferation and DNA synthesis of ECs with a significant decrease in the percentage of cells in the G0/G1 phase and a significant increase in the S phase.15 Chorioallantoic membrane assay verified that FA increased VEGF and platelet-derived growth factor expressions in ECs and upregulated the amount of hypoxic-induced factor-1α mRNA.16 Electrospinning is a process by which ultrafine fibers with diameters in the micrometer down to nanometer range can be fabricated. Electrospun fibers have attracted a great deal of attention, especially in biomedical fields, as scaffolds for tissue engineering and drug delivery because of their similar skeletal structure to extracellular matrix (ECM), flexible variation of morphology, interconnected pores, and high specific surface area, and have been shown to be effective for local and sustained delivery of bioactive signals.17 Zhang et al. assessed the responses of ECs and SMCs on electrospun silk fibroin

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EXPERIMENTAL SECTION Materials and Cells. Poly(ethylene glycol)-poly(DL-lactide) (PELA, Mw = 42.3 kDa, Mw/Mn = 1.23) was prepared by bulk ring-opening polymerization of lactide/poly(ethylene glycol) using stannous chloride as the initiator.24 AT and FA of over 99% purity were purchased from National Institutes for the Control of Pharmaceutical and Biological Products (Beijing, China). All the electrophoresis reagents, bovine serum albumin (BSA), and dimethyl sulfoxide (DMSO) were procured from Sigma (St. Louis, MO). Protein molecular weight marker and RIPA lysis buffer were from Beyotime Institute of Biotechnology (Shanghai, China). Rabbit anti-human antibodies of collagen I, collagen IV, laminin, α-smooth muscle actin (αSMA), and β-actin; rabbit anti-mouse antibodies of CD31, collagen IV, and α-SMA; and goat anti-rabbit IgG−fluorescein isothiocyanate (FITC), IgG−horseradish peroxidase (HRP), and 3,3′-diaminobenzidine (DAB) developer were purchased from Boster Bioengineering Co., Ltd. (Wuhan, China). All other chemicals were analytical grade and received from Changzheng Regents Company (Chengdu, China) unless otherwise indicated. ECs were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein, and expanded in complete medium 199 containing 20% fetal calf serum (Gibco BRL).25 Human umbilical artery SMCs were obtained by outgrowth of cells from discarded pieces of umbilical artery media, and cultured in DMEM/F12 medium with 10% fetal bovine serum (Gibco BRL).26 ECs and SMCs of the third generation were used in the following evaluations. 2395

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Effect of the Concentration and Feeding Ratio of AT and FA on the Cell Viability. The viabilities of ECs and SMCs were determined after treatment with AT and FA of different concentrations and different ratios between them. Briefly, EC or SMC suspensions were seeded in 96-well tissue culture plates (TCP) with a cell density of 2 × 103 cells/well. After 24 h incubation to allow cell attachment, AT and FA solution in culture medium was added into each well. The total concentration of AT and FA was set between 0 and 100 μg/ mL, and the concentration ratio of AT to FA was between 10/0 and 5/5. After 5 days of incubation, the culture medium was removed, followed by the addition of 100 μL of fresh culture medium and 10 μL of CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) into each well. After incubation for 2 h according to the reagent instruction, an aliquot (100 μL) of incubated medium was pipetted into another 96-well TCP and the absorbance of each well was measured at 450 nm using a μQuant microplate spectrophotometer (Elx-800, Bio-Tek Instrument Inc., Winooski, VT). The same volumes of culture medium and CCK-8 reagent were incubated without cell as the background. All experiments were performed with n = 6. Preparation of Electrospun Fibers. Electrospun PELA fibers with AT and FA loadings were prepared as described elsewhere.27 Briefly, polymers and drugs were codissolved in DMSO, which was transferred into a 5 mL syringe attached to a blunt metal needle as the nozzle. A high voltage difference of 20 kV was applied between the nozzle and a grounded collector through a high voltage statitron (Tianjing High Voltage Power Supply Co., Tianjing, China), and a steady flow at 2.0 mL/h out of the nozzle was controlled by a microinject pump (Zhejiang University Medical Instrument Co., Hangzhou, China). Fibers were subsequently spun on the aluminum foil wrapped on a rotating mandrel, and the fibrous mats collected were vacuum-dried overnight to remove residual solvents and then stored at 4 °C. Electrospun fibers with the encapsulation of AT and FA with different ratios were prepared, and fibers F7/3 indicated the encapsulation of AT and FA with the weight ratio of 7/3. Electrospun fibers mats without drug inoculation were also prepared as F0. Characterization of Drug-Loaded Fibrous Mats. The morphology of electrospun fibers was investigated by a scanning electron microscope (SEM, FEI Quanta 200, The Netherlands) equipped with a field-emission gun (20 kV) and a Robinson detector after 2 min of gold coating to minimize the charging effect. The fiber diameter was measured from SEM images as described previously.27 The loading amount and encapsulation efficiency of AT and FA were determined after extraction from fibers. Briefly, a known amount of fibers (ca. 2 mg) was dissolved in 500 μL of chloroform and extracted three times with 2.5 mL of pH 7.4 phosphate buffered saline (PBS). The amount of AT in the extracted solution was measured by using a fluorescence spectrophotometer (Hitachi F-7000, Japan) with the excitation wavelength of 310 nm and the emission wavelength of 376 nm,28 and that of FA by using a UV spectrophotometer (Shimadzu UV2550, Japan) at the wavelength of 310 nm.29 The actual concentration was obtained using a standard curve from known concentrations of AT and FA solutions. The drug loading amount, determined in triplicate for at least five different batches and given as a percentage, indicated the amount (in milligrams) of drug encapsulated per 100 mg of electrospun fibers. The encapsulation efficiency indicated the percentage of drug

encapsulated with respect to the total amount used for the fiber preparation. In Vitro Release Profile of AT and FA from Electrospun Fibers. The drug-loaded fibers were punched into small squares with a total mass of around 10 mg, and were sterilized by electron-beam irradiation using a linear accelerator (Precise, Elekta, Crawley, U.K.) with a total dose of 80 cGy. The fibrous mats were immersed, in triplicate, in 10 mL of PBS, and the suspension was kept in a thermostated shaking water bath that was maintained at 37 °C and 100 cycles/min. At predetermined time intervals, 1.0 mL of the release buffer was removed for analysis and fresh PBS was added for continuing incubation. The concentrations of AT and FA in the release media were detected as described above. Cell Culture on Drug-Loaded Fibrous Mats. The growth behaviors of primary ECs and SMCs were determined on fibrous mats F9/1, F7/3, and F5/5, compared with corresponding drug-infiltrated fibrous mats. Briefly, before cell seeding 12 mm diameter disks (about 10 mg) were punched from fibrous mats, fixed by a cell-culture ring with 10 mm of internal diameter as designed by Zhu et al.,30 and sterilized. Drug-infiltrated fibrous mats were prepared by dropping culture media containing AT and FA with the weight ratios of 9/1, 7/3, and 5/5 onto F0 fibrous mats, which were named as F09/1, F07/3, and F05/5, respectively. The fibrous mats were placed into 48-well TCP, and 100 μL EC or SMC suspensions with a cell density of 5 × 104 cells/mL were seeded, which were maintained at 37 °C in a humidified atmosphere for 4 h to make cells diffuse into and adhere to the scaffold before the addition of 900 μL of culture medium into each well. Characterization of Cell Viability on Fibrous Mats. For the cell viability test, cell-loaded fibrous mats were rinsed, moved to another 48-well TCP, and immersed with 200 μL of fresh culture medium in each well. Then 20 μL of CCK-8 reagent was added into each well, and the cell viability was assayed as described above. The cell attachment was examined 4 h after cell seeding, and the cell viability test was repeated on days 1, 3, and 7 after incubation. Cells were removed from fibrous scaffolds by trypsinization after 7 days of culture, and an almost complete removal was observed by SEM. Another batch of cells was seeded on the recovered fibrous mats and incubated for an additional 7 days, and the cell depletion and inoculation processes were repeated for the total culture time of 28 days. All experiments were performed with n = 6. Characterization of ECM Secretion of Cells on Fibrous Mats. The cell-loaded fibrous mats were retrieved after 7 days of incubation for the evaluation of cell morphology and ECM secretion. For the morphology observation, cell-loaded fibrous mats were washed with PBS twice, and then fixed with 4% glutaraldehyde for 2 h at 4 °C. Following three rinses with distilled water, the samples were dehydrated through a series of graded ethanol solutions and then freeze-dried. Dry constructs were sputter coated with gold and observed by SEM. The secretion of collagen IV and laminin by ECs and that of collagen I and α-SMA by SMCs was observed by immunofluorescent staining. Briefly, cell-loaded fibrous mats were washed with PBS three times and then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 solution in PBS. After blocking by incubation with 2% BSA for 30 min at 37 °C, the fibrous mats were incubated with rabbit anti-human antibodies of collagen I, collagen IV, laminin, and α-SMA for 2 h at 37 °C. After being washed three times with PBS, samples were incubated with goat anti-rabbit IgG−FITC 2396

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in the dark for 1 h at 37 °C. The scaffolds were washed three times with PBS, mounted, and observed using a confocal laser scanning microscope (CLSM, Leica TCS SP2, Germany) with the excitation and transmission wavelength of 488 and 535 nm, respectively. Western blot was used to determine collagen IV secretion by ECs, and α-SMA by SMCs. Briefly, cells on the fibrous mats were homogenized in RIPA lysis buffer, and total proteins of the cell lysate were determined by BCA protein assay kit. The cell lysate was then mixed with loading buffer (40 mM TrisHCl, 1% SDS, 50 mM DTT, 7.5% glycerol, 0.003% bromophenol blue) and then subjected to electrophoresis on 10% SDS−PAGE gel at 100 V, followed by transfer to PVDF membrane (Millipore Corp., Bedford, MA). After blocking with 5% BSA for 2 h at room temperature, the membrane was washed and incubated with rabbit anti-human antibodies of collagen IV and α-SMA for 1 h at room temperature and kept at 4 °C overnight. Membranes were then washed three times with PBS containing 0.05% Tween 20 and reacted with goat anti-rabbit IgG−HRP for 1 h. Antigen−antibody complexes were visualized by DAB developer. Expression of β-actin was used as protein loading control. Subcutaneous Implantation of Fibrous Mats in Rats. The angiogenesis promotion was assessed after subcutaneous implantation of drug-loaded fibrous mats F9/1, F7/3, F5/5, using drug-infiltrated mats F09/1, F07/3, F05/5, and empty mats F0 as control. Male SD rats were from Sichuan Dashuo Biotech Inc. (Chengdu, China), weighing 100 to 120 g, and all animal protocols were approved by the University Animal Care and Use Committee. Briefly, the rats were anesthetized by intraperitoneal injection of pentobarbital sodium at a dosage of 30 mg/kg. All visible hair was removed from the graft site with 8% Na2S aqueous solution, and subcutaneous pockets were made on both sides on the back of rats. The sterilized electrospun fibrous mats were rolled into a cylindrical shape, and triple samples of each fibrous group were put into the subcutaneous pockets before suturing the incisions. Characterization of Blood Vessel Formation. The scaffolds with surrounding tissue were retrieved after 1, 2, and 4 weeks of implantation. Approximately half of each explant was frozen and cut into 4 μm section for standard hematoxylin−eosin (HE) staining to evaluate the fiber residual, inflammatory reaction, and vessel formation. The other portion of the explant was fixed in formalin, paraffin-embedded, and sectioned into 4 μm slices for immunohistochemical (IHC) analyses of CD31, collagen IV, and α-SMA. Briefly, sections were incubated with 3% H2O2 for 10 min to inactivate the endogenous peroxidase, and put into 10 mM citrate buffer solution (pH 6.0) and heated in a microwave oven to recover antigen. Nonspecific binding sites were blocked with 5% BSA in Tris-buffered saline for 20 min, and the sections were incubated with primary antibodies at 4 °C overnight. Then biotinylated secondary antibodies were incubated with the slides at 37 °C for 30 min, followed by incubation with streptavidin−HRP for 20 min. The slides were incubated with a DAB−H2O2 solution to visualize antibody binding sites. After counterstaining for 1 min with hematoxylin, the sections were dehydrated with sequential ethanol for sealing, and observed with a light microscope (Nikon Eclipse E400, Japan). At least 10 individual microscopic images were randomly selected from 3 different IHC slides of each sample. Collagen IV and α-SMA-positive vessels with morphological circumference of staining of one or more cells were counted, while positive staining with single dot

was not counted as capillary vessels.31 The blood vessel density was normalized to tissue area (number of vessels/mm2). Statistics Analysis. The results are reported as mean ± standard deviation (SD). Whenever appropriate, two-tailed Student’s t-test was used to discern the statistical difference between groups. A probability value (p) of less than 0.05 was considered to be statistically significant.

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RESULTS AND DISCUSSION Effect of the Concentration and Feeding Ratio of AT and FA on the Cell Viability. Until now there have been few reports to clarify the effect of AT and FA treatment on the behaviors of vascular cells. Wang et al. indicated that the proliferation of endothelial cell line ECV304 increased notably in a dose-dependent manner on FA concentrations without significant cytotoxicity, while the cell viability decreased when the FA concentration reached 100 μg/mL.15 Zhao et al. showed that FA inhibited the protein and DNA synthesis of vascular SMCs by 61% at a concentration of 200 μg/mL.32 In the current study the viabilities of primary ECs and SMCs were evaluated after incubation with AT and FA of different concentrations and feeding ratios. As shown in Figure 1, the

Figure 1. The viabilities of ECs and SMCs after incubation with AT and FA of different concentrations and feeding ratios, compared with culture media without drug inoculation (dotted lines) (n = 6).

relative cell viability was dependent on the concentrations of AT and FA, and an optimal concentration of around 50 μg/mL was determined for both ECs and SMCs. The combined treatment with AT and FA with the ratios from 9/1 to 5/5 led to significantly higher cell viability than those with individual drug (p < 0.05), indicating the synergism of the combined treatment on both ECs and SMCs. Compared with those without drug treatment, the cell viability indicated over 2-fold higher for ECs after treatment with AT and FA with the ratio 7/3 and the total drug concentration of 20−50 μg/mL. The highest viability of SMCs was also obtained for AT and FA treatment with the ratio of 7/3 among all the dosing regimens. 2397

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Characterization of Drug-Loaded Fibers. Figure 2 shows typical SEM morphologies of drug-loaded fibers,

Figure 2. SEM image of electrospun fibers F7/3 with AT and FA loadings.

displaying a highly porous and randomly interconnected structure. Drug-loaded fibers indicated bead-free smooth morphology with the diameters of 0.65 ± 0.15 μm and average drug-loading efficiency of 95%. Efficient encapsulation of therapeutics into fibers was one of the advantages of the electrospinning process,17 and there was no significant difference in the size and drug-loading efficiency among fibers with different drug loadings. Drug Release Profiles from Electrospun Fibers. Figure 3 summarizes the cumulative release profiles of AT and FA from fibers during 30 days of incubation. All the fibers indicated similar release profiles containing three phases: an initial burst release during initial 24 h and a sustained and quick release for around 1 week, followed by a gradual release for about 3 weeks. Figure 3a shows the release profiles from fibers containing 0.25%, 0.5%, and 1.0% of AT, indicating that fibers with lower drug-loaded amount exhibited lower amount of initial burst release and total release during the incubation period. There were around 15.7%, 25.1%, and 31.6% of initial release during 1 day incubation, and around 59.3%, 70.9%, and 79.4% of accumulated release within 30 days for fibers with AT loadings of 0.25%, 0.5%, and 1.0%, respectively. When the amount of drug entrapment was increased, drug molecules may aggregate more on the fiber surface, leading to an even larger initial burst release. Figure 3b shows the release behaviors of FA, indicating a slightly quicker release than AT from fibers of the same drug loading amount. Such a result was consistent with the higher hydrophilicity and lower molecular weight of FA than those of AT, facilitating the escape of FA from fibers. The burst release during 1 day incubation was around 23.9%, 28.5%, and 33.3%, and the total release during 30 days was around 75.6%, 81.1%, and 87.7% from fibers with drug loadings of 0.25%, 0.5%, and 1.0%, respectively. There is a noteworthy point that the amount of gradual release after the initial burst release indicated no significant difference for fibers with different drug loadings. As shown in Figure 3a and Figure 3b, the amount of gradual release was around 46% for AT-loaded fibers, while that of FAloaded fibers was around 53%. Figure 3c shows the release of AT and FA from fibers with loadings of both drugs. There was around 80.7% of AT and 80.8% of FA release from fibers containing 1.0% of AT and

Figure 3. Percent release of (a) AT and (b) FA from electrospun fibers with drug loadings of 0.25%, 0.5%, and 1.0% after incubation in PBS, pH 7.4 at 37 °C (n = 3). (c) Percent release of AT and FA from electrospun fibers with loadings of 1.0% AT and 0.5% FA after incubation in PBS, pH 7.4 at 37 °C (n = 3).

0.5% of FA during 30 days of incubation, indicating release profiles similar to those with inoculation of individual drug. It indicated that there were sufficient sites within fibers available for drug loading, and no interference of the coloading of AT and FA on the drug release rate. These above results made it possible to regulate the amount of drug release by controlling the drug loading mass and ratios in fibrous mats. Considering the effect of AT/FA ratios on the cell viability (Figure 1), codelivery of AT and FA with different ratios by F9/1, F7/3, and F5/5 fibers with the total loading amount of 1.0% was used to clarify the effects on the behaviors of ECs and SMCs and the vascular formation after subcutaneous implantation. Cell Growth Behaviors on Fibrous Mats. The viabilities of ECs and SMCs were determined on fibrous mats with encapsulated AT and FA, compared with drug-infiltrated fibrous mat and empty fibers. As shown in Figure 4a, drugencapsulated fibers with different ratios of AT and FA displayed different effects on cell viabilities. Compared with F0 fibers, F7/ 3 fibers led to around 2.5-fold and 2.2-fold higher cell viabilities of ECs and SMCs, respectively. There were significant differences in the cell viabilities between drug-encapsulated and drug-infiltrated fibrous mats (p < 0.05), which may be due to the existence of an optimal drug concentration for cell growth (Figure 1), and the sustained availability of drugs released from fibers (Figure 3). As shown in Figure 4a, the viabilities of both ECs and SMCs were significantly higher after incubation with F7/3 fibers than those of F9/1 and F5/5 fibers (p < 0.05), which was consistent with the different synergistic 2398

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Figure 4. (a) The viabilities of ECs and SMCs after incubation for 7 days with drug-encapsulated fibers (F9/1, F7/3, and F5/5) and drug-infiltrated fibers (F09/1, F07/3, and F05/5), compared with empty fibers F0 (n = 6; *: p < 0.05). (b) The viabilities of ECs and SMCs after incubation with F7/ 3 and F07/3 fibers for 28 days, compared with empty fibers F0 (n = 6; *: p < 0.05). (c) SEM morphologies of ECs and SMCs after incubation on F7/ 3 fibers for 7 days. Bars represent 20 μm.

which were the proper cellular phenotypes for functional vascular grafts.33 ECM Secretion of Cells on Fibrous Mats. The ECM secretion is an important step for generation of functional tissues. For example, the production of a basal lamina is an important role of ECs, which plays a central role in organizing and establishing all membranes and indicates substantial functions in the microvessel formations.34 The substantial function of vascular SMCs is to maintain the integrity and function of mature blood vessels through expressing smooth muscle-specific variants of cytoskeletal and contractile proteins, including α-SMA, myosin, collagen I, and elastin.35 In the present study, the secretion of collagen IV and laminin by ECs and that of collagen I and α-SMA by SMCs were evaluated by immunofluorescent staining after 7 days of culture on drugencapsulated fibers. Figure 5a demonstrated strong ECM expressions for both ECs and SMCs, with slabstone-like and oriented contractile feature, respectively. Figure 5b summarizes Western blotting results of collagen IV and α-SMA expressions, using β-actin as protein loading control. As shown in the band densities, the amount of collagen IV and α-SMA extracted from cells treated with F7/3 fibers was significantly higher than those from other fibers, which reflected results similar to the cell viability tests (Figure 4). It was indicated that a continuous and slow release of AT and FA at effective ratios stimulated the growth of both ECs and SMCs and enhanced the ECM expression. Tissue Growth into Fibrous Mats after Subcutaneous Implantation. The fibrous mats with surrounding tissue were explanted at different intervals after subcutaneous implantation, and HE staining on tissue sections was employed to examine the inflammation reaction, tissue infiltration, and formation of blood vessels. Acute inflammation was developed after the implantation of fibrous scaffold, but began to regress after implantation for 3−5 days.36 As shown in Figure 6a, the accumulation of lymphocytes and macrophages was detected after 1 week implantation, and most inflammatory cells

actions after combined treatment with AT and FA of different ratios (Figure 1). The cell attachment and viability were determined after incubation with F7/3 and F07/3 compared with F0 fibers, and the results are summarized in Figure 4b. The attachment of ECs and SMCs on F7/3 and F07/3 indicated profiles similar to that of F0, indicating the drug release from fibers and the drug presence in the media had no effect on the cell attachment efficiency. The relative cell viability indicated a slight increase during 7 days of culture of ECs on F07/3 fibers, while no significant difference was found for SMCs in the meantime (p > 0.05). However, F7/3 fibers showed continuous increases in the relative cell viabilities, which were significantly higher than those of F07/3 fibers after 3 days of incubation (p < 0.05). The gradual drug release from fibers was supposed to stimulate cell growth continuously. In the present study, ECs and SMCs were removed from fibrous scaffolds by trypsinization after 7 days, and another batch of cells were seeded on the recovered fibrous mats and incubated for an additional 7 days. The cell depletion and seeding processes were repeated, and the relative cell viability was determined after 14, 21, and 28 days of incubation. As shown in Figure 4b, there was a slight decrease in the relative cell viability compared with that on day 7 after incubation, but no significant difference was found after 14, 21, and 28 days of incubation (p > 0.05). Compared with those on F0 fibers, there was over 1.6-fold higher cell viability of both ECs and SMCs on F/3 fibers during 28 days of incubation, indicating the capabilities of drug-encapsulated fibers to extend the availability of AT and FA at effective ratios within local tissue environment. The cell morphology and distribution within the fibrous mats were observed by SEM. As shown in Figure 4c, cells were tightly attached on and spread over multiple fibers, stretched very well, likely due to contact guidance of these fibers. Similar to those after culture on TCP, ECs and SMCs indicated flat membrane and contractile features on fibrous mat, respectively, 2399

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implantation of empty fibers and drug-encapsulated and druginfiltrated fibrous mats, indicating that the inflammation was caused by implantation of fibers other than drugs released from fibers. Figure 6b shows the tissue infiltration into fibrous scaffolds and fiber residuals after implantation. At week 1 after implantation, the boundaries between fibers and infiltrated cells were well-defined. After 2 weeks of implantation, the fibrous scaffolds were fragmented, overstaffed with the penetration of body fluids and occupied by tissue ingrowth although the border existed. With the speed of scaffold degradation and tissue regeneration, few fiber fragments were observed in histological sections at week 4 after operation, and grown tissues were presented throughout the samples. It was indicated that fibers were degraded and absorbed along with cell migration and tissue ingrowth, leading to the recruitment of vascular cells and arrangement into a circular structure to form blood vessels. As shown in Figure 6c, vessel-like structures were arranged in new tissues around fibers after 1 week of implantation, and blood vessels with filled with red blood cells were generated after 2 and 4 weeks. It was demonstrated that the generated vessels were integrated with native vasculature. Blood Vessel Formation after Subcutaneous Implantation of Fibrous Mats. Mature blood vessels are characterized by a layer of ECs surrounded by SMCs and pericytes in the walls, and the ECM deposition provides a structural framework essential for the functional properties of vessel walls.37 CD31 was used primarily to demonstrate the presence of ECs, and the deposition of collagen IV indicated a similar distribution of ECs.38 Figure 7a and Figure 7b show the typical images of CD31 and collagen IV staining on tissue explants after implantation of F9/1, F7/3, F5/5, and F0 fibers, indicating the recruitment of ECs into a circular morphology in all the tissue sections. The collagen IV-positive staining was used to manually determine the total number of blood vessels,39 and Figure 7c summarizes the quantitative counts. At week 1 the implantation of F0 fibers led to formation of blood vessels with a density of around 60 vessels/mm2. There were 80−90 vessels/mm2 after implantation of drug-encapsulated and drug-infiltrated fibrous mats, and no significant difference was found among them (p > 0.05). The inflammatory response after fiber implantation and fiber degradation may stimulate the endothelial cell migration, and the presence of AT and FA and degradation products of fibers should induce strong proliferation on numerous inflammatory cells, high metabolic rate, and low oxygen content in regenerated tissues, which were supposed to promote vasculogenesis.33 As shown in Figure 7c, the vessel densities indicated no significant difference after 2 and 4 weeks of implantation of F09/1, F07/3, F05/5, and F0 fibers compared with those at week 1 (p > 0.05). However, the implantation of F9/1, F7/3, and F5/5 fibers for 2 weeks led to significantly higher vessel densities than those of week 1 (p < 0.05), at 158 ± 13, 197 ± 16, and 168 ± 12 vessels/mm2, respectively. There were slight decreases in the vessel densities at week 4 after implantation of F9/1, F7/3, and F5/5 fibers, at 140 ± 11, 180 ± 13, and 142 ± 11 vessels/mm2, respectively. The α-SMA secretion is usually used to study the level of maturation of the newly formed blood vessels.40 Figure 8a shows the typical IHC staining images for α-SMA expression of tissue explants, indicating the existence of α-SMA-secreting cells, such as SMCs and pericytes in the walls of newly formed

Figure 5. (a) Immunofluorescent staining images of collagen IV and laminin expressions by ECs, and collagen I and α-SMA by SMCs after incubation for 7 days with F7/3 fibers. Bars represent 20 μm. (b) Western blotting assay of collagen IV expressions by ECs and α-SMA by SMCs after incubation for 7 days with F9/1, F7/3, and F5/5 fibers, compared with empty fibers F0. Total proteins were prepared from cell lysate, and β-actin was used as protein loading control.

Figure 6. (a) HE staining of inflammatory reaction after implantation of F7/3 fibers for 1, 2, and 4 weeks. White arrow heads indicate lymphocyte, and black ones macrophages. (b) Fiber residuals and cell infiltration observed by HE staining after implantation of F7/3 fibers for 1, 2, and 4 weeks. M represents the fibrous matrices, and T the infiltrated tissues. Arrow heads indicate the fiber residuals, and dotted lines the interface of cell infiltration. (c) HE staining for blood vessel formation after implantation of F7/3 fibers for 1, 2, and 4 weeks. Arrow heads indicate vessels with filled red blood cells. Bars represent 50 μm.

distributed at the margin between layers of the scaffold. The inflammatory reaction became weakened rapidly, and HE staining of tissues retrieved after 4 weeks of implantation indicated few inflammatory cells remaining. There was no significant difference in the inflammation reactions after 2400

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Figure 8. (a) IHC staining images for α-SMA expression of tissue explants treated with F9/1, F7/3, and F5/5 fibers and empty fibers F0 for 4 weeks. Arrow heads indicate α-SMA-positive vessels with morphological circumference. Bars represent 200 μm. (c) Quantitative counts of α-SMA-positive vessels and normalized to tissue area (no. of vessels/mm2) after implantation of drug-encapsulated fibers (F9/1, F7/3, and F5/5), drug-infiltrated fibers (F09/1, F07/3, and F05/5), and empty fibers F0 for 1, 2, and 4 weeks. *: p < 0.05.

time point (p > 0.05). However, compared with those at week 1, the vessel densities indicated significant increases at week 2, at 121 ± 10, 150 ± 12, and 131 ± 9 vessels/mm2 for F9/1, F7/ 3, and F5/5 fibers, respectively. Moreover, there were slight increases in the vessel densities at week 4 after implantation of F9/1, F7/3, and F5/5 fibers. As shown in Figure 8b, F7/3 fibers led to significantly higher densities of mature blood vessels than F9/1 and F5/5 fibers after 2 and 4 weeks of implantation (p < 0.05). Blood Vessel Regeneration by Fibrous Scaffolds with Codelivery of AT and FA. The promotion of blood vessel initiation and growth plays an important role in realizing therapeutic vascularization and regeneration of functional tissues. Although a lot of growth factors and plasmids could facilitate angiogenesis, their unstable activity, expensive prices, and toxicity have limited their application. Angelica sinensis and astragalus membranaceus are the commonly used TCM for enriching the blood, and the two medicines are made up as a prescription “Danggui Buxue Tang”. Yang et al. investigated the effect of the different ratios of angelica sinensis and astragalus membranaceus injections on angiogenesis, indicating that combination treatment obviously enhanced the proliferation and migration of vascular ECs and stimulated the angiogenesis of a chick embryo chorioallantoic membrane.41 Li et al. indicated that the decoction of astragalus membranaceus and angelica sinensis enhanced the synthesis of DNA and mitosis, and increased VEGF gene expression in cultured ECs.42 The main ingredients of astragalus membranaceus and angelica

Figure 7. IHC staining images for CD31 (a) and collagen IV expressions (b) of tissue explants treated with F9/1, F7/3, and F5/5 fibers and empty fibers F0 for 4 weeks. Arrow heads indicate CD31 or collagen IV-positive vessels with morphological circumference. Bars represent 200 μm. (c) Quantitative counts of collagen IV-positive vessels and normalized to tissue area (no. of vessels/mm2) after implantation of drug-encapsulated fibers (F9/1, F7/3, and F5/5), drug-infiltrated fibers (F09/1, F07/3, and F05/5) and empty fibers F0 for 1, 2, and 4 weeks. *: p < 0.05.

blood vessels. The quantitative counts of mature microvessel densities after 1, 2, and 4 weeks of implantation are shown in Figure 8b. The positively stained vessels were around 40 vessels/mm2 after 1 week implantation of F0 fibers, while slightly higher densities of 50−60 vessels/mm2 were obtained after implantation of drug-encapsulated and drug-infiltrated fibers. The implantation of drug-infiltrated fibers led to an increase in the vessel densities of around 80 vessels/mm2 at week 2, followed by a slight decease to around 68 vessels/mm2 at week 4. There was no significant difference in the densities of mature vessels among F09/1, F07/3, and F05/5 fibers at each 2401

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CONCLUSIONS Electrospun fibers with the loadings of AT and FA were evaluated in the current study to promote the generation of blood vessels. The cell viability after AT and FA treatment was dose-dependent with an optimal concentration of around 50 μg/mL, and the most significant synergistic effect was shown for the combined treatment with AT/FA ratio of 7/3 for both ECs and SMCs. The in vitro release study showed that the amount of AT and FA release can be regulated by their loading amount and ratios in electrospun fibers. Compared with other dosing regimens, F7/3 fibers indicated significantly higher cell viability and ECM secretion during 28 days of incubation with both ECs and SMCs. The localized and sustained codelivery of AT and FA led to significantly higher densities of blood vessels and mature vessels than drug-infiltrated fibers after subcutaneous implantation, and the most significant angiogenesis promotion was demonstrated for F7/3 fibers. Although there are many challenges that need to be addressed, the integration of synergistic effect of TCMs into electrospun fibrous scaffolds should provide clinical relevance for getting fully vascularized in engineered tissues and regenerating promptly blood vessel substitutes.

sinensis are AT and FA, respectively, and their angiogenesis promotion has been identified through increasing the proliferation of vascular cells and promoting the expressions of angiogenic growth factors.13 In the current study the viabilities of ECs and SMCs were initially evaluated after incubation with AT and FA of different concentrations and feeding ratios, and the most significant synergistic effect was demonstrated for the combined treatment with AT and FA with the ratio of 7/3 (Figure 1). Moreover, the synergistic effect of AT and FA on vascular cells was integrated into electrospun fibers to provide abundant and sustained factors required to initiate vascularization and bring it to maturity. Previous reports indicated that AT and FA dampened the cell proliferation at high doses and had short half-lives in vivo,21 which made it necessary to establish a sustained release of AT and FA. As shown in the in vitro release study, the coloading of AT and FA into electrospun fibers showed no interference on the release rate of individual drug (Figure 3c). In addition, the amount of gradual release after the initial burst release indicated no significant difference for fibers with different drug loadings (Figure 3a,b). Therefore, the amount of AT and FA release could be regulated by their loading amount and ratios in electrospun fibers, which made it applicable to clarify the synergistic effect on angiogenesis promotion of electrospun fibers loaded with AT and FA of different ratios. Compared with other dosing regimens, F7/3 fibers indicated significantly higher cell viability (Figure 4a) and ECM secretion (Figure 5) for both ECs and SMCs, and induced significantly higher densities of vascular structures after implantation (Figures 7 and 8). As shown in Figure 7c, a slight decrease in the vessel densities was detected after 4 weeks of implantation of either drug-loaded or drug-infiltrated fibers compared with those at week 2, indicating the newly formed capillaries needed to be made mature to protect them from degradation. While a decrease in the densities of mature vessels was observed at week 4 after implantation of F09/1, F07/3, and F05/5 fibers compared with those at week 2, an increase in the vessel densities was detected for F9/1, F7/3, and F5/5 fibers (Figure 8c). It was indicated that the sustained release of AT and FA promoted the maturation of newly formed vessels. TCMs are becoming accepted worldwide for their effectiveness, weak side effects, and safety.10 Previously pDNA polyplexes were encapsulated into electrospun fibers to induce sufficient protein expression to promote the regeneration of mature blood vessels. Both the attachment capability and viability of ECs after 7 days of incubation on fibrous mats with pDNA polyplexes encapsulated were around 75% compared with those of empty fibers.20 However, around 2-fold higher cell viability was shown for both ECs and SMCs after incubation on F7/3 fibers than that of F0 fibers (Figure 4b). In addition, compared with empty fibers, the implantation of fibrous mats with pDNA polyplexes included induced strong inflammation both between fibrous layers and at the interface of fibrous mats with penetrated tissues, and necrotic tissue was observed followed by scar formation.20 However, a weak inflammatory reaction was found after implantation of fibers with AT and FA loadings, and the inflammation was caused by implantation of fibers other than drugs released from fibers (Figure 6a). To summarize, electrospun fibers with codelivery of AT and FA revealed a clinically applicable and efficient way to enhance the growth of mature blood vessels.

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AUTHOR INFORMATION

Corresponding Author

*School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, P.R. China. Phone: +8628-87634068. Fax: +8628-87634649. E-mail: xhli@swjtu. edu.cn. Author Contributions §

H.W. and Y.Z. contributed equally to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (51073130 and 21274117), Specialized Research Fund for the Doctoral Program of Higher Education (20120184110004), and National Scientific and Technical Supporting Programs (2012BAI17B06).

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