Article pubs.acs.org/journal/abseba
Silk Fibroin-Based Scaffolds with Controlled Delivery Order of VEGF and BDNF for Cavernous Nerve Regeneration Yaopeng Zhang,*,†,‡ Jianwen Huang,†,§ Li Huang,‡ Qiangqiang Liu,‡ Huili Shao,‡ Xuechao Hu,‡ and Lujie Song*,§ ‡
State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, P. R. China § Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, P. R. China S Supporting Information *
ABSTRACT: To investigate the synergistic effect of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) on cavernous nerve regeneration, two different aligned scaffolds consisting of coaxial electrospun silk fibers were prepared by switching the position of the two factors in either core or shell domain. The order and release rate of the dual factors delivery were relatively different because of the distinct location of two factors in coaxial fibers. An in vitro assay showed that the inner-VEGF/outerBDNF scaffolds could more obviously accelerate Schwann cells growth, proliferation and spreading owing to the rapid release of BDNF. However, in vivo scaffold implantation demonstrated that the inner-BDNF/outer-VEGF scaffolds significantly facilitated more angiogenesis, and promoted more nerve regeneration based on great benefit of angiogenesis. Results showed that the reasonable dual-delivery order of VEGF and BDNF from scaffolds could enhance synergistic effect of the factors and promote cavernous nerve regeneration. KEYWORDS: coaxial electrospinning, silk fibroin scaffolds, dual-delivery order, cavernous nerve regeneration
1. INTRODUCTION Erectile dysfunction (ED) is a significant problem for many men after radical prostatectomy (RP) due to prostate carcinoma. Previous studies have reported that about 20% of men following RP experience postoperative ED, which is due to a combination of cavernous nerve (CN) injuries, such as traction, transection, and electrocautery injury.1−4 Thus, it is very necessary to repair and reconstruct CN following RP. Because many tissue-engineered synthetic materials and natural polymers were used for the repair of peripheral nerve injuries,5−8 the tissue-engineered materials may also be alternatives for CN regeneration. Silk fibroin from Bombyx mori, an attractive biomaterial, has good biocompatibility, controlled degradability, nontoxicity, and versatile processability in different material formats.9,10 Thus, silk fibroin has a wide range of applications in the biomedical fields such as bone,11,12 skin,13,14 low urinary tract,15,16 and vascular17,18 repair. Silk fibroin-based conduits also achieved considerable success for nerve regeneration.7,19−23 Electrospun fiber-based scaffolds with the natural extracellular matrix (ECM) structures showed an increased cellular proliferation and exchange of nutrient/waste.24−26 Compare to electrospun fiber scaffolds27 with random structures, those with aligned structures are capable of guiding neurite extension and Schwann cell (SC) migration.28 Therefore, regenerated silk fibroin (RSF) aqueous solution was used © XXXX American Chemical Society
to electrospin the scaffolds consisting of aligned microfibers for CN repair and reconstruction. Moreover, the organic-solventfree spinning process and scaffolds are environmentally friendly and avoid potentials risk for cells. The artificial constructs loaded with neurotrophic factors, such as nerve growth factor (NGF), glial cell line derived neurotrophic factor (GDNF) and neurotrophin NT-3, brainderived neurotrophic factor (BDNF), are evidently effective to improve peripheral nerve regeneration.29−33 Previous studies have demonstrated that the combined therapy of vascular endothelial growth factor (VEGF) and BDNF promoted the neural regeneration and produced the most robust neurite outgrowth than the independent use of BDNF or VEGF.34,35 Our recent in vivo and in vitro experiments also demonstrated that dual factor-loaded RSF scaffolds simply mixed with BDNF and VEGF were more effective for peripheral nerve treatment than the bare RSF scaffolds.36 However, the synergistic effect of BDNF and VEGF has not been well understood. To boost the synergistic effect, it is necessary to investigate how the release order, dose, and time of the double factors affect the nerve regeneration efficiency. Received: July 29, 2016 Accepted: September 6, 2016
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DOI: 10.1021/acsbiomaterials.6b00436 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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scaffolds in this study was about 70 μm, which can be controlled by adjusting the electrospinning time. The coaxial electrospinning nozzle with an inner tube and an outer tube was used. The inner tube of the nozzle has an inner diameter of 0.45 mm and an outer diameter of 0.80 mm, while the outer tube has an inner diameter of 2.00 mm and an outer diameter of 3.20 mm. The shell dope and core dope were fed through a syringe by a syringe pump (KDS210P, KD scientific, Inc., USA) at a flow rate of 1.2 and 0.3 mL/h, respectively. The applied voltage was 20 kV from a high-voltage power supply (ES60P, Gamma High Voltage Research, Inc. USA) and the distance between nozzle and collector was 10 cm. The previous studies have demonstrated that aligned fibers of silk fibroin or other polymers could guide cell orientation to enhance nerve regeneration.38−40 To make coaxial fibers well-organized, a roller covered with aluminum foil at a rotating rate of 2000 rpm was served as collector. The tubular scaffolds on the foil were then cut and removed from the roller. After the aluminum foil was peeled off, the flat sheet scaffolds were obtained. The as-spun scaffolds were treated by water vapor annealing at 37 °C and 90% relative humidity for 36 h to make them insoluble in water.41 The densities of 20 and 39 wt % RSF aqueous solutions were about 1.08 and 1.13 mg/mL by measuring the weight and the volume of the solutions. On the basis of an assumption that all of the solvent evaporated after the fiber formation, factor loading capacity in the core and shell of coaxial fibers was calculated according to the following equation
In this study, two types of aligned silk-based scaffolds were prepared by simply switching the position of BDNF and VEGF for either core or shell domain using coaxial electrospinning to achieve time-programmed dual release formulation. To further amplify release order, dose, and time of double factors, we intentionally loaded a smaller dose into the core domain of fibers than the shell domain. It is more obvious to understand the effects of the delivery order of the dual factors on SCs’ growth, spreading on the scaffolds and CN regeneration in the rat model with CN injury, respectively.
2. MATERIALS AND METHODS 2.1. Materials. Cocoons of B. mori were purchased from Tongxiang, China. Cellulose dialysis membranes (Molecular weight cutoff 14000 ± 2000 D) were acquired from Yuanju Co., Ltd. (Shanghai, China). BDNF and VEGF were obtained from Gibco Life Technologies Co., USA. Human BDNF and VEGF ELISA kit were purchased from Raybiotech, Inc. USA. Trypsin, Dulbecco Modied Eagle’s Medium (DMEM) and penicillin-streptomycin were obtained from Jinuo Biomedical Technology co., Ltd. (Hangzhou, China). Mouse Schwann cells at passage 3 were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. The purity of Schwann cells was estimated to be greater than 95% using flow cytometer. All other chemicals of analytical grade were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.2. Preparation of Electrospun RSF Scaffolds and Factors Loading. B. mori cocoons were boiled twice in 0.5 wt % Na2CO3 solution for 30 min to separate the silk fibroin from the sericin glue. The degummed silk was then dissolved using 9.0 M LiBr aqueous solution (10:1 w/v) at 40 °C for 2 h. After diluted and centrifuged at 3500 rpm for 10 min, the supernatant silk solution was dialyzed against deionized water with the cellulose dialysis membrane for 3 days to remove LiBr. When silk solution was concentrated to 20 wt %, 2 μg BDNF was mixed with 16 mL silk solution and the blended solution was then concentrated to 39 wt % by forced air flow as the shell dope for coaxial electrospinning. As to the core dope for coaxial electrospinning, 0.5 μg/mL VEGF- bovine serum albumin (BSA) aqueous solution was used. The above scaffolds were designated as IVOB (inner-VEGF/outer-BDNF) and listed in Table 1. The two
core loading capacity (ng/mg) = Q cCfc/(D39Q s39 wt%)
shell loading capacity(ng/mg) = 2 × 103ng/(D20 ·16 mL·20 wt%) (2) where Qc and Qs are the flow rates of the core and shell dope, the Cfc is the factor concentration of the core dope. D20 and D39 are the densities of the 20 and 39 wt % RSF aqueous solutions. 2.3. Morphology Observation of Scaffolds. Scanning electron microscopy (SEM, JEOL JSM-1435600LV, Japan) was used to observe the morphology of RSF scaffolds at a voltage of 10 kV. The scaffolds were sputter-coated with gold before observation. The average diameter and angle distribution (with respect to their major axis) of the fibers were measured using image analysis software (UTHSCSA ImageTool 2.0).42,43 For each sample, 100 fibers from SEM images were randomly counted. 2.4. Mechanical Properties. The rectangular specimens (35 × 5 mm2) cut from the scaffolds were tested along the direction parallel or perpendicular to the aligned fibers, respectively. Instron tensile tester (Instron 5969 material testing machine, USA) with 5 N load cell was used. The thickness of each specimen was the average of 10 measurements taken by a CH-1-S thickness gauge (Shanghai Liuling Instruments Co., Shanghai, China). Tensile properties were performed at a drawing speed of 3 mm/min and each group was measured with at least 10 samples. To test the suture retention strength, the sample was held in the lower grip and threaded with a suture (5−0 polyglactin, Ethicon, USA) 2 mm from its upper edge. The ends of the suture were fixed to the upper grip. The suture retention was defined as the peak force obtained during this procedure.42 2.5. In Vitro Release of Factors from Scaffolds. In vitro release of BDNF and VEGF from scaffolds IVOB and scaffolds IBOV were measured using an enzyme-linked immunosorbent assay (ELISA). Samples of 100 mg each group (n = 3) were placed in a tube
Table 1. Dual-Growth Factors Loaded in the Scaffolds with Coaxial Fibers samples
inner layer
outer layer
VEGF loading amount (ng/mg)
BDNF loading amount (ng/mg)
IVOB IBOV
VEGF BDNF
BDNF VEGF
2.84 × 10−1 5.80 × 10−1
5.80 × 10−1 2.84 × 10−1
(1)
dopes were then switched to load VEGF and BDNF in outer layer and inner layer of coaxial fiber, respectively. The resultant sample was designated as IBOV (inner-BDNF/outer-VEGF). Schematic drawing of the preparation process of scaffolds with aligned RSF fibers by coaxial electrospinning was shown in Figure 1. The flat scaffolds (width × height: 10 cm × 10 cm) consisting of aligned microfibers with core−sheath structures were prepared using previously descried procedures.37 The average thickness of the
Figure 1. Schematic drawing of the preparation process of scaffolds with aligned RSF fibers by coaxial electrospinning. B
DOI: 10.1021/acsbiomaterials.6b00436 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering containing phosphate-buffered saline (PBS) and then incubated at 37 °C for up to 16 days. At the predetermined time points, 400 μL of PBS was extracted and a same-volume fresh one was then replenished. The cumulative release was calculated on the basis of the released amounts at each point. 2.6. Cell Culture and In Vitro Examination. SCs were cultured in the Dulbecco’s modified Eagle’s medium (DMEM, Gibco Inc.) with 10% (v/v) fetal bovine serum (FBS, HyClone Inc.), 100 units/mL of penicillin, and streptomycin (Gibco Inc.). L-Glutamine was also contained in the DMEM. For cell seeding, disks with 1.4 cm diameter were cut from two scaffolds and placed in 24-well plates. All the samples were sterilized with 75% alcohol for 2 h. Two types of scaffolds loaded with dual factors were used as experiment groups, while glass coverslips and tissue-culture polystyrene (TCPS) were used as control samples. Because coverslips and TCPS were negative control groups for the adhesion and proliferation of SCs, coverslips and TCPS precoated with poly-L-lysine (molecular weight range 70− 150 kDa, Sigma-Aldrich, Inc.) were also used as control groups to evaluate SCs adhesion and proliferation. The cell viability of SCs on the different substrates was evaluated by MTT at 1, 3, 5, and 7 days. Each sample was repeated three times to perform the evaluation. Before SCs were seeded, immunofluorescent (IF) staining was performed using monoclonal antibodies (anti-S100β protein) against the SCs to identify the SCs. For the spreading and cytoskeleton of the SCs on the two scaffolds, SCs were seeded with concentration of 2 × 104 cells for 3 days. The scaffolds were then fixed with 2.5% paraformaldehyde at 4 °C for 2 h, following by dehydration with a series of graded ethanol solutions. The proliferation of SCs on scaffolds then was observed by SEM. The cytoskeleton of SCs on scaffolds observation was performed by rhodamine phalloidin staining. After wash, fixation and permeation, the stained samples were observed at a wavelength of 561 nm using laser scanning confocal microscopy (LSCM) (TCS SP5, Leica Microsystems Inc., Germany). Meanwhile, 3D cytoskeletal morphology images of SCs were acquired from Imaris 6.2 software (Bitplane Co., Switzerland). 2.7. Surgical Procedure and In Vivo Examination. The whole animal experimental protocol was approved by the Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, before study initiation. Thirty 12-week-old, male, Sprague−Dawley rats were assigned to 3 groups. Group RSF-neat received a CN repair with RSF scaffolds without addition of growth factors (n = 10). Group IVOB received a CN repair with scaffolds IVOB (n = 10) and Group IBOV received a CN repair with scaffolds IBOV (n = 10). All surgeries were performed by the same surgeon, and CN injury model was established according to our precious work.4 Briefly, the surgical procedure was performed under anesthesia with sodium pentobarbital (40 mg/kg, i.p.), and a lower midline abdominal incision exposed the prostate gland and seminal vesicle. The major pelvic ganglion (MPG), the inflow (pelvic nerve and hypogastric nerve) and outflow (CN) were identified on one side of the prostate under an operating microscope (×10). A CN gap with a mean length of 5 mm was created by transection, and RSF-neat scaffolds with a length of 9 mm and a width of 2 mm were sutured using 10-zero absorbable polyglactin sutures between two CN ends. The long axis of the scaffolds was parallel with their aligned fibers (Figure 2). IVOB and IBOV scaffolds were sutured in the same manner in the group IVOB and group IBOV, respectively. All animals were administered prophylactic antibiotics (5 mg/kg/d ofloxacin) for 3 days postoperatively. After 4 and 8 weeks of implantation, rats were euthanized by excessive amounts of anesthetic and implanted scaffolds were harvested for immunohistochemical (IHC) analyses. For IHC analysis, the nerve (NF-200 protein) and vessel markers (von Willebrand factor) were detected using the monoclonal antibodies: anti-NF-200 protein and antivon Willebrand factor, respectively. The sections on each group sample were observed by an optical microscopy (DS-Ri1, Nikon, Tokyo, Japan). 2.8. Statistical Analysis. All experiments were performed at least in triplicate, and data were expressed as mean values ± standard deviation. Statistical analysis of data was performed by one-way
Figure 2. Macroscopic views illustrating RSF scaffolds implantation in rat model. (a) Exposed major pelvic ganglion (MPG, black arrow), pelvic nerve (PN, green arrow), hypogastric nerve (HN, white arrow) and cavernous nerve (CN, yellow arrow); (b) establishing CN gap (arrow) with a length of 5 mm using scissors and (c) suturing schematic of RSF scaffolds and two CN ends. analysis of variance (ANOVA) and Bonferroni’s test. A value of p < 0.05 was considered significant and indicated in the figures as *(p < 0.05).
3. RESULTS AND DISCUSSION 3.1. Morphological Characterization and Mechanical Properties. The SEM micrographs of the scaffolds consisting of coaxial electrospun fibers were shown in Figure 3. The average diameter of the fibers was 2.5 ± 0.7 μm. It was reported
Figure 3. (a) SEM images of electrospun RSF scaffolds with aligned fibers, (b) corresponding diameter distribution of the RSF fibers, and (c) the histograms of the fiber angle distribution. AD and SD are the abbreviations of average diameter and standard deviation, respectively. C
DOI: 10.1021/acsbiomaterials.6b00436 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering that diameters of fibers were at a proper range for neurite outgrowth and Schwann cell migration. Small fiber diameters around 200 nm had negative effect on the neurite outgrowth and Schwann cell migration, while microfibers with diameters greater than 30 μm were also reported as inhibitory to the neurite outgrowth and Schwann cell migration.28,44 The quantitative analysis of fiber alignment was used by statistical analysis of the angles between the fibers and their major axis. Figure 3c reveals that most of the fibers in the aligned scaffolds formed angles from 0° to 30° with respect to the axis. Hence, it can be considered that most fibers arranged along a certain direction. Moreover, the uneven and ribbon-shaped silk fibers were observed, likely due to the slow water evaporation from fiber surface.45 It can be seen that many fibers merged together. We can consider that the electrospun fibers were physically cross-linked by themselves, which is good for the performance enhancement of the mechanical properties of the scaffolds. Although the combination of fiber may reduce the pore size and porosity of the scaffolds, the large average pore size about 7.5 μm and porosity over 61% of the scaffolds are still sufficient for the cell growth, proliferation, and nutrition exchange. The method of porosity determination can be found in the Supporting Information. As shown in Figure 4, the electrospun RSF scaffolds with aligned fibers exhibited anisotropic tensile properties. In the
Figure 5. Factor-loading schematic and cumulative release profiles of (a, a′) VEGF and (b, b′) BDNF from different domains of (A) IVOB and (B) IBOV in PBS (pH 7.0) at 37 °C.
ng/mg in the core and shell domains of IVOB and IBOV fibers, respectively. The BDNF loading amount in the core and shell domains of the fibers was the same as VEGF. In other words, the theoretically complete releases of the shell and core factors from 100 mg scaffolds were 5.8× 104 and 2.84 × 104 pg, repectively. For IVOB in Figure 5A, the cumulative releases of BDNF and VEGF close to 5.4 × 103 and 2.0 × 103 pg (9.3% and 7.0%), respectively. For IBOV in Figure 5B, the cumulative releases of BDNF and VEGF were close to 1.3 × 103 and 6.5 × 103 pg (4.6% and 11.2%), respectively. It can be known that the factor in the shell released faster than the same factor in the core. Moreover, VEGF has a faster release rate than BDNF even in the same domain. This may be ascribed to the strong ionic interactions between RSF (negative charge, pI = 4.3) and BDNF (positive charge, pI = 9.1) in contrast to VEGF (pI = 8.5), or their different degradation rate.46,47 In a word, the two scaffolds can slowly release the dual factors and control the release order, dose and time of the dual factors effectively. According to our previous results,37 the RSF scaffolds annealed in water vapor may exhibit higher release profiles than those immersed in ethanol. 3.3. In Vitro Examination. Cell viability of SCs on the different substrates by MTT on 1, 3, 5 7 day after SCs seeding was shown in Figure 6. The OD value of SCs on all the substrates increased gradually from day 1 to day 7. It is known that poly-L-lysine coated surfaces could significantly enhance the adhesion and proliferation of SCs. Figure 6 shows that the SCs seeded on both type of scaffolds after 3 days exhibit faster growth than all control groups, including coverslips, TCPS, poly-L-lysine coated coverslips and poly-L-lysine coated TCPS. After 5 days, the proliferation rate of the SCs cultured on IVOB was significantly higher than that on IBOV (p < 0.05). After 7 days, the spreading and proliferation conditions of the SCs on different surfaces show the following order: poly-L-lysine coated TCPS > poly-L-lysine coated coverslips > IVOB > TCPS > IBOV > coverslips. This indicated that enough rapidly released BDNF with bioactivity from the shell domain of IVOB could effectively promote the growth of SCs. This was consistent with Koda’s results:48 BDNF significantly decreased the number of dead SCs; the number of transplanted cells into contused rat
Figure 4. Typical stress−strain curves of the electrospun RSF scaffolds stretched along the directions (a) parallel and (b) perpendicular to the aligned fibers.
parallel direction to the aligned fibers, the RSF scaffolds have a strong tensile strength of about 9.8 MPa and a relatively low elongation at break of 1.4%. In the perpendicular direction to the aligned fibers, however, the scaffolds showed a weak tensile strength of 2.1 MPa and an elongation at break of 1.2%. The suture retentions of the scaffolds in the parallel and vertical directions to fibers were 0.27 ± 0.03 N and 0.69 ± 0.11 N, respectively. In our work, the suturing of the scaffolds and the two CN ends demonstrated appropriate mechanical properties of the scaffolds for in vivo applications. According to our previous work,37 the tensile properties and suture retention can be further improved by increasing the thickness of the scaffolds and building composite scaffolds with bladder acellular matrix graft. 3.2. Release Behaviors of BDNF and VEGF from Scaffolds. Figure 5 shows the release behaviors of BDNF and VEGF from different domains of coaxial electrospun microfibers in PBS (pH 7.0) during 16 days. The initial burst release can be obviously observed at first 4 d, followed by a stable release up to 16 days. The release profiles of the factors in different locations were unique and an initial burst of the factors in the core was highly suppressed. As shown in Table 1, the VEGF loading amount was 2.84 × 10−1 and 5.80 × 10−1 D
DOI: 10.1021/acsbiomaterials.6b00436 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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higher dose, SCs on sample IVOB had more healthy morphology and exhibited faster spread rate than on sample IBOV. This is consistent with the MTT assay results. Meanwhile, most SCs spread along the orientation of fibers, which is of great help for nerve regeneration.49 Figure 8 shows the rhodamine phalloidin staining morphology of SCs cultured on the two scaffolds for 3 days. It was noticed that the most spindle morphology of SCs appeared in the LSCM 2D images. The morphology of SCs cultured on IVOB was not significantly different from that of the cells grown on IBOV in LSCM 2D images. However, in the LSCM 3D images, it was obvious that SCs on IVOB maintained better morphology as spindles than the cells on IBOV. To some extent, the adequate BDNF from the shell domain of IBOV actually improved SCs growth. In addition, the depth of z dimension of the LSCM 3D images is about 30 μm, proving that SCs could penetrate into the scaffolds. 3.4. In Vivo Examination. To detect the vascularization and nerve regeneration simultaneously, IHC staining was adopted on the endothelial maker (vWF) and the innervation marker (NF-200 protein) in the three types of scaffolds at each time point, respectively. The obvious positive areas (brown) were respectively observed on the vWF (yellow arrows in Figure 9a) and on NF-200 protein (yellow arrows in Figure 10a), demonstrating vascularization and nerve regeneration in the scaffolds after implantation. The quantitative analyses of the images are shown in Figure 9b and Figure 10b. In the three types of scaffolds, both vessel and innervation regeneration present a significant increase at 8 weeks than that at 4 weeks. Furthermore, the numbers of vessels induced by IBOV with VEGF in sheath domain was significantly higher than that occurring in the IVOB with VEGF in core domain at two time points. This is because VEGF is a main facilitator in the process of angiogenesis.50 VEGF loaded in the shell domain of IBOV produces rapid release to encourage more initial vascularization at the implantation site. It is expected that the scaffolds with growth factors improve more vessel regeneration compared to the neat scaffolds, which is consistent with our previous study.36 Although the proliferation rate of the SCs cultured on IVOB was higher than that on IBOV in vitro, more nerves grew on the retrieved IBOV than IVOB at each time point in the rat model. Because of the beneficial role of angiogenesis in tissue regeneration, BDNF from IBOV could maximize its function in promoting innervation regeneration with the presence of adequate blood vessels. It could be considered that the silkbased scaffolds with controlled release order, dose, and time of VEGF and BDNF could efficiently promote vessels and nerves regeneration. The synergistic effect of VEGF and BDNF made the functional silk scaffolds suitable for CN regeneration as well as further uses in other repair and reconstruction. Normally, scaffolds for tissue engineering should have suitable biodegradability after implantation in body.51 It was quite obvious that silk-based scaffolds can gradually degrade in the CN gap rat model. In the images of IHC staining, it can be seen that the scaffolds (denoted by the asterisk) are not integrated and exhibit a small amount of degradation after 4 weeks of implantation. After 8 weeks, only few residual scaffolds can be found. It indicates that the RSF scaffolds can provide microenvironment for regeneration of vessels and nerves at prior period. And when enough vessels and nerves regenerated or tissues were repaired, the silk scaffolds reveal extensive degradation. Unlike other synthetic polymer scaffolds, no reoperation is needed to remove the silk scaffolds.
Figure 6. Cell viability of SCs after 1, 3, 5, and 7 days of cell cultured on different substrates. The asterisk (*) indicates significant difference between the groups at p < 0.05. IVOB, IBOV, and TCPS stand for inner-VEGF/outer-BDNF scaffolds, inner-BDNF/outer-VEGF scaffolds, and tissue-culture polystyrene, respectively. The sample number is 3.
spinal cord in the BDNF-treated group was significantly larger than that in the nontreated group. Figure S1a shows the spindle-shaped morphology of the SCs on TCPS. The SCs with an estimated purity greater than 95% were further identified by IF staining using anti-S100β protein (Figure S1b). The SEM and LCSM morphologies of SCs cultured on the two type scaffolds were shown in Figures 7 and
Figure 7. SEM images of SCs cultured for 3 days on scaffolds of (a) IVOB and (b) IBOV.
8. From SEM micrographs, it can be seen that SCs attached and spread well on the scaffolds, indicating the two scaffolds had good cytobiocompatibility. Due to the first released BDNF with
Figure 8. LSCM 2D (first line) and 3D (second line) images of SCs cultured for 3 days on scaffolds of (a, a′) IVOB, and (b, b′) IBOV. E
DOI: 10.1021/acsbiomaterials.6b00436 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 9. (a) IHC staining to evaluate vessel in the retrieved scaffolds of RSF-neat, IVOB, and IBOV at 4 and 8 weeks after implantation. The asterisk (*) denotes the scaffolds fragments and the yellow arrows denote vessels. Bar: 100 μm. (b) Image analyses of the vessel densities in a. The asterisk (*) in b denotes significant difference among the groups at each time point (P < 0.05).
Figure 10. (a) IHC staining to evaluate innervation in the retrieved scaffolds of RSF-neat, IVOB, and IBOV at 4 and 8 weeks after implantation. The asterisk (*) denotes the scaffolds fragments and the yellow arrows denote neuronal lineages. Bar: 200 μm. (b) Image analyses of NF-200 positive contents of cavernous nerve fibers in a. The asterisk (*) in (b) denotes significantly difference among the groups at each time point (P < 0.05).
4. CONCLUSIONS Two types of silk fibroin-based scaffolds for CN regeneration were successfully prepared by switching the position of BDNF and VEGF for either core or shell domain using coaxial electrospinning. The delivery order and doses of dual factors were effectively controlled by the different factor-loading ways. The scaffolds with a core of RSF-VEGF and a sheath of RSFBDNF promoted the spreading and proliferation of SCs in vitro than the counterparts with a core of RSF-BDNF and a sheath of RSF-VEGF. On the contrary, vascularization and nerve regeneration were considerably fast in the scaffolds with a core of RSF-BDNF and a sheath of RSF-VEGF at the same period of time in a rat model. Reasonable release order could improve the synergistic effect of the dual factors. BDNF further enhanced the CN regeneration process in the presence of enough blood vessels. In a word, silk fibroin-based scaffolds consisting of coaxial fibers are a potential controlled delivery carrier for growth factors and the inner-BDNF/outer-VEGF scaffolds have improved performance for CN regeneration. The effect of the dual-factor release order on the vascularization and nerve regeneration may be also promising and useful for other
tissues repair and reconstruction. To extend the in vivo application of the scaffolds for nerve regeneration, it is also possible to convert the flat scaffolds to a nerve guidance conduit (NGC) by reeling and sealing process.52
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00436. Method of porosity determination, morphology and identification of SCs before seeding on scaffolds (PDF)
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AUTHOR INFORMATION
Corresponding Authors
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DOI: 10.1021/acsbiomaterials.6b00436 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Author Contributions †
Y.Z. and J.H. contributed equally and should be considered cofirst authors Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is sponsored by the National Natural Science Foundation of China (21274018, 21674018, 81671451, 81600524), “Shuguang Program” supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (15SG30), DHU Distinguished Young Professor Program (A201302), the Fundamental Research Funds for the Central Universities, the Programme of Introducing Talents of Discipline to Universities (111-2-04) and State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University (LK1408).
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