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Alg composite scaffold loaded NGF for spinal cord injury repair
Materials Science and Engineering C 76 (2017) 81–87
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A bridging SF/Alg composite scaffold loaded NGF for spinal cord injury repair Genlong Jiao a,1, Yongqin Pan b,1, Cunchuang Wang b,⁎, ZhaoXia Li c,⁎, Zhizhong Li a, Rui Guo d,e,⁎⁎ a
Department of Orthopedic, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China Department of General Surgery, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China Department of Rheumatology, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China d Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China e Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China b c
a r t i c l e
i n f o
Article history: Received 22 June 2016 Received in revised form 19 December 2016 Accepted 21 February 2017 Available online 22 February 2017 Keywords: Silk fibroin Scaffold Alginate microspheres Nerve growth factor Spinal cord injury
a b s t r a c t Neurons loss and axons degeneration after spinal cord injury (SCI) gradually give rise to result in functional motor and sensory impairment. A bridging biomaterial scaffold that allows the axons to grow through has been investigated for the repair of injured spinal cord. In this study, we introduced a silk fibroin (SF)-based neurobridge as scaffold enriched with/without nerve growth factor (NGF) that can be utilized as a therapeutic approach for spinal cord repair. NGF released from alginate (Alg) microspheres on SF scaffold (SF/Alg composites scaffolds) to the central lesion site of SCI significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons. This optimal multi-disciplinary approach of combining biomaterials, controlled-release microspheres and neurotrophic factors offers a promising treatment for the injured spinal cord. © 2017 Published by Elsevier B.V.
1. Introduction Spinal cord injury (SCI) is one kind of serious trauma which could cause loss of neurons and degeneration of axons, and further gradually result in the loss of sensory and motor functions [1]. Because of the severity of SCI, there is no effective treatment to completely cure at present. Incidence of SCI presents an increasing tendency, so the discovery of new therapeutic strategies has become a clinically urgent need nowadays. Current treatment to SCI includes surgery to decompress and stabilize the injury, prevention of secondary complications, management of any that do occur, and rehabilitation [1,2]. However, the microenvironment at the spinal cord injury site is rather complicated, and more than one process needs to be regulated in order for axonal regrowth to occur [3]. Not only should hindering factors, such as gliosis or inflammation, be minimized, but the controlled release of necessary neurotrophic factors should be
⁎ Corresponding authors. ⁎⁎ Correspondence to: R. Guo, Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China. E-mail addresses: [email protected] (C. Wang), [email protected] (Z. Li), [email protected] (R. Guo). 1 The authors contributed equally to this work.
sustained; so the theoretical approach to repairing an injured spinal cord is to regenerate damaged axons through the site of injury [4]. A bridging biomaterial construct that allows the axons to grow through has been investigated for the repair of injured spinal cord [5]. Due to the hostility of the microenvironment in the lesion, multiple conditions need to be fulfilled to achieve improved functional recovery. An attractive strategy for repairing injured spinal cord is to design of a new bioactive scaffold that can bridge the gap of the lesion as contact guidance for axonal growth and act as a vehicle to deliver neurotrophic factors in order to modify the microenvironment [6,7]. Silk fibroin (SF), a non-toxic, non-immunogenic structural protein [8], has been successfully explored for the nerve repair research [9]. Scaffolds fabricated from SF delivering neurotrophin (NT)-3 were applied in the treatment of a rat SCI model, and the study showed improved axonal growth [10]. In another study, bi-layer SF scaffolds and small intestinal submucosa matrices could support bladder tissue regeneration in SCI rat's model [11]. Nerve growth factor (NGF) is an important member of the neurotrophin family and an important regulator of neural survival, development, function, and plasticity [12]. It has been indicated that NGF shows neuroprotective effects and improves the recovery of SCI [13–15]. NGF was selected due to its stable and high binding properties, as well as the regulation of neural survival, development, function, and plasticity. Although NGF is important for their mitotic and differential properties for endogenous neural progenitors and their ability to
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accelerate neovascularisation, the application of NGF was limited in the clinic because of its short half-life, rapid dilution in the body [16]. So it is important to localize and sustain release of NGF to the nerve injury site. Alginate (Alg) is one of the most commonly used natural biopolymer, its biodegradability and drug release properties could be regulated by composition, sequence, G-block length, and molecular weight [17–19]. At present, biodegradable Alg microspheres were widely applied in the drug controlled release system [20–22]. It has been reported that Alg microspheres were used as an injectable vehicle capable of filling cavities in the SCI, and of providing the substrate for axon attachment and re-growth [23,24]. In order to overcome the technical bottlenecks in the treatment of SCI, we designed a promising therapeutic strategy combining biomaterials, controlled-release microspheres and neurotrophic factors for the injured spinal cord: SF-based neurobridge as the scaffold and Alg-microsphere as the controlled-release delivery carrier. NGF were loaded on the SF/Alg composite scaffolds to promote neurons outgrowth and regeneration in the SCI rats model, and the combined therapeutic effects of SF/Alg/NGF composite scaffold for SCI repair were evaluated.
2. Materials and methods
other chemicals were of analytical reagent quality and used without further processing. 2.2. Preparation of the SF/Alg/NGF composite scaffolds NGF-loaded Alg microspheres were prepared according to the literature [17,25,26]. Briefly, sodium Alg was dissolved in purified water to a concentration of 2.0% (w/v) with overnight stirring at room temperature. NGF (100 μg/mL), and then added to the Alg solution by shaking gently for 10 min. The NGF-loaded Alg solution was then injected into a 50 mM CaCl 2 solution to form microspheres via a syringe and a high direct-current (DC) voltage. The microspheres were hardened for 15 min and then washed for three cycles using purified water. The final concentration of the SF solution was ~ 4% (w/v), which was determined gravimetrically after freeze-drying for 24 h to remove any remaining solvent. The SF/Alg/NGF scaffold was produced by mixing 1 mL of 4% (w/v) SF solution and 5 mg of Alg/NGF, and then pouring the solution (100 μL) into a 96-well plate and freeze-drying for 24 h. The NGF/ SF scaffold was fabricated by mixing 1 mL of 4% (w/v) SF solution and 4 μL of NGF solution (0.5 μg/μL) and then pouring the mixture (100 μL) into a 96-well and freeze-drying for 24 h. Pure SF scaffolds were also prepared.
2.1. Materials 2.3. Scanning electron microscopy Calcium alginate and pentobarbital was obtained from Sigma (USA). B. mori silkworm cocoons used in the experiment were kindly donated by Sijia Min from Zhejiang University. NGF was purchased from R&D Systems (USA). Dulbecco's-modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). PC12 cells were donated by the Bio-Engineering Institute of the Jinan University. Cell Counting Kit-8 (CCK-8, Beyotime, China). All
Scaffolds of the various SF/Alg/NGF blends were cut out and inspected before and after methanol treatment under a stereomicroscope (Leica Microsystems, Switzerland). Methanol treated samples were also observed by scanning electron microscopy (SEM). SEM samples were coated with platinum prior to evaluation with a LEO 1530 GEMINI scanning electron microscope (Zeiss, Cambridge, UK).
Fig. 1. SEM images of Alg microsphere (A), SF scaffolds (B) and SF/Alg scaffolds (C).
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intervals, 1 mL supernatant was removed for analysis and replenished with 1 mL fresh buffer for continuing incubation. The concentration of NGF released from the scaffolds was quantified by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's guidelines (R&D Systems, USA). All release experiments were carried out in triplicate.
2.6. Bioactivity of the released NGF
Fig. 2. FTIR spectra of Alg, SF and SF/Alg.
Histological sections (see below) were inspected by light microscopy (IX71, Olympus, Japan). 2.4. Structure analysis The infrared (IR) spectra of SF, Alg and SF/Alg were obtained using a Fourier transform infrared (FTIR) spectrometer (Vertex 70; Bruker, Billerica, MA). The IR spectra in the absorbance mode were recorded using a diamond crystal plate and obtained in the spectral region 400–4000 cm − 1 with a resolution of 4 cm − 1 and 20 scans per sample. Raman spectra were recorded on a Bruker MultiRam FT-Raman spectrometer equipped with a cooled Ge-diode detector. The excitation source was a Nd3t-YAG laser (785 nm) in the backscattering (180°) configuration. The focused laser beam diameter was about 100 mm, the spectral resolution 4 cm− 1, and the laser power at the sample about 100 mW. 2.5. In vitro release of NGF The in vitro release of SF/NGF and SF/Alg/NGF scaffolds was evaluated in samples immersed in 50 mL phosphate-buffered saline (PBS), pH 7.4, at 37 °C with shaking at 100 rpm. At predetermined time
The bioactivity of NGF released from SF/NGF and SF/Alg/NGF samples in vitro was examined by determining its ability to stimulate the proliferation of PC12 cells. Released NGF samples collected at day 1, 7 and 14 from SF/NGF and SF/Alg/NGF in 1 mL medium (same with the cell culture medium) were used. PC12 cells were cultured in DMEM supplemented with heat-inactivated 10% FBS, 5% horse serum, and antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin) and incubated in a humidified incubator at 37 °C with 5% CO2. Cells were seeded on 96-well plates (4000 cells/well) at 37 °C, 5% CO2. After cell attachment (24 h), the culture medium was then discarded and an additional 100 μL/well fresh complete DMEM was added. After incubation for another 24 h, 10 μL of the collected released NGF samples from SF/NGF and SF/Alg/NGF medium at different sampling times (at day 1, 7 and 14) with the same NGF concentration of 1.5 μg/mL adjusted by fresh medium, was added to each well, respectively. Positive control condition was also studied. For the positive control condition, 10 μL of fresh release medium with 15 ng standard NGF was added. After 48 h incubation, cell viability was determined using a CCK-8 assay (Beyotime, China) with a fluorescence plate reader, according to the manufacturer's instructions.
2.7. Animals 72 healthy adult female Wistar rats (240–260 g) were obtained from the Experimental Animal Center of Guangdong (Guangzhou, China). The rats were housed in a specific pathogen-free room at a constant temperature of 25 °C and humidity of 45%. The present study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Science (CAMS, China).
Fig. 3. Raman spectra of SF and SF/Alg.
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2.8. Animal model establishment, grouping and implantation treatment The SCI SF was established in accordance with the modified Allen's method. The rats were anesthetized with 400 mg/kg chloral hydrate via intraperitoneal injection. A laminectomy was subsequently performed on the entire spinous process, the vertebral plate of T9 and part of the vertebral plate of T8 and T10, to expose the dorsal (posterior) surface of the spinal cord. The exposed spinal cord at the T9 level was vulnerated with a 10 g weight dropped from a height of 2.5 cm (vulnerating energy, 25 g/cm). Following injury, the rats were randomly divided into three groups: the SF group (n = 24), SF/NGF group (n = 24), SF/Alg/NGF group (n = 24). The prepared scaffolds (SF, SF/NGF and SF/Alg/NGF) were implanted into the wounded site of the rats with SCI respectively. 2.9. Behavior and histological detection At week two, four, six and eight following transplantation, an inclined plane test was conducted and Basso, Beattie, Bresnahan (BBB) locomotor rating scale values were obtained for the rats in the control and transplantation groups. Samples collected from the rats at week four and eight were stained with hematoxylin-eosin (HE) dyes, to examine the histological changes. 2.10. Immunofluorescence Serial 5-μm paraffin-embedded sections of rats' spinal cord were prepared to detect the protein expression of MAP2 and NeuN in each experimental group. After dewaxing, sections were incubated in 3% H2O2 for 15 min and then in blocking solution for 1 h at 37 °C. Subsequently, the sections were incubated at 4 °C overnight with the following primary antibodies: anti-MAP2 (Sigma, 1:500), anti-NeuN (Abcam, 1:500). After washed three times with PBS, sections were incubated with DyLight® secondary antibodies (Abcam, 1:1000) for 2 h at 37 °C. The results were photographed through a light microscope (IX71, Olympus, Japan), and micrographs were analyzed using Image-Pro plus 6.0 software. 2.11. Statistical analysis All experimental data were analyzed with the repeated one-way analysis of variance (ANOVA), which was followed by a Newman– Keuls post hoc test for multiple comparisons using SPSS 17.0 software (SPSS Inc., USA). p b 0.05 was considered to be statistically significant.
Fig. 4. In vitro NGF release profiles from SF/NGF and SF/Alg/NGF scaffolds.
3.2. FTIR and Raman analysis The infrared (IR) spectra of SF, Alg and SF/Alg were performed and shown in Fig. 2, the presence of SF could be observed in SF/Alg composite; characteristic SF peaks at 676, 1064, 1528 and 1668 cm−1 were observed [27]. The Raman spectrum (Fig. 3) of SF showed amide I band at 1668–1640 cm−1 and a complex amide III band with components at 1272 and 1253–1245 cm−1 [28]. In the Raman spectra of the SF scaffolds some bands are definitely ascribable to side-chain vibrations of specific amino acids. Among them, the Raman bands of Tyr at 1615, 1211, 1174, 854, 830, and 644 cm−1 appeared with higher intensity in SF. The bands of SF (1668, 1335 and 854 cm−1) were observed in SF/ Alg scaffolds.
3.3. In vitro NGF release NGF release studies were carried out to evaluate the ability of Alg to control the release of the loaded drug. As shown in Fig. 4, in SF/NGF scaffolds, N65% of the NGF was rapidly released within 4 h. After 72 h, almost all NGF loaded in the SF/NGF scaffold was released. The drug release rate was the slowest in the SF/Alg/NGF scaffolds; NGF was released gradually from the SF/Alg/NGF scaffolds over 240 h, only about 8.23% of the NGF released in the first 4 h period.
3. Results 3.1. Characterisation of Alg microspheres and composite scaffolds From the SEM micrographs of Fig. 1A, all the Alg microspheres had a spherical shape and that their surfaces were pitted with holes. The mean dry diameter of the microsphere was 198 ± 5.9 μm. The dry microspheres tended to swell when immersed in water and became transparent; the average diameter microsphere after swelling in water was 401 ± 7.8 μm. The Alg microspheres fabricated using a water-in-oil process had a porous structure, with an average swelling ratio of 7.8 ± 0.8, which suggested that these microspheres could be applied as drug carriers. Fig. 1B shows the SEM images of the SF scaffold, the pore distribution was uniform, with an average pore size of 75–90 μm. The porosity of the SF scaffold was 96.2 ± 0.7%. A typical SEM image of the SF/Alg scaffold is shown in Fig. 1C. Compared with Fig. 1B, the SF/Alg scaffold exhibited an even distribution of pores, with an average pore size of 30–45 μm (porosity: 83.8 ± 0.9%). Alg microspheres were observed in the pore walls of the SF scaffold and were well distributed; holes were also observed in the microsphere surface.
Fig. 5. Biological activities of NGF released from SF/NGF and SF/Alg/NGF scaffolds. (*p b 0.05 vs. SF/NGF).
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3.6. Histological observation Pathological changes of the spinal cord samples from the experimental groups at 4 W and 8 W after treatment were shown in Fig. 7. The neurons in the spinal cord of the SF group were shrunken or had pale homogenous cytoplasm. Obvious improvement on tissue repairment were observed in both SF/NGF and SF/Alg/NGF group at 4 W after treatment compared with the SCI group; and at 8 W, the two groups showed more ameliorative effects with less necrosis and karyopyknosis. These results further strengthened that SF/Alg/NGF administration has a neuroprotective effect and improves the SCI recovery. 3.7. Promoting neurons growth Fig. 6. The BBB locomotion scores of all experimental groups from 2 weeks to 8 weeks after treatment (*p b 0.05 vs. SCI group).
3.4. Bioactivity of released NGF in the cell proliferation assay To examine the biological activity of released NGF, a CCK-8 assay was used to evaluate the PC12 cells proliferation. From the data in Fig. 5, the cell viability of SF/Alg/NGF group at day 1, 7 and 14 showed no significant difference by comparing numbers between the different time points. Moreover, compared with SF/ NGF, SF/Alg/NGF promoted cell proliferation and viability (p b 0.05 vs. SF/NGF), suggesting that Alg incorporation retained the bioactivity of NGF.
Immunofluorescence detection was used to examine the expression of the neuronal specific marker after transplantation. Microtubuleassociated protein 2 (MAP2) is a neuronal protein that regulates the structure and stability of microtubules, neuronal morphogenesis, cytoskeleton dynamics, and organelle trafficking in axons and dendrites [29]. Neuronal nuclei (NeuN) is a nuclear protein expressed in most post-mitotic neurons of the central and peripheral nervous systems [30]. From the results of the immunofluorescence (Figs. 8–9), more MAP2- and NeuN-positive neurons were observed in the spinal cord of the SF/Alg/NGF and SF/Alg/NGF groups at 4 W after treatment compared with the SCI group; and at 8 W, the two groups showed more significant effects. 4. Discussion
3.5. Animal behavior function The open field locomotor function was assessed using the BBB scoring method at 2 W, 4 W, 6 W, 8 W after scaffolds transplantation. As shown in Fig. 6, all the rats displayed complete hind limb paralysis after SCI. In the following 2–8 weeks, locomotor performance substantially improved. Beginning in the 2nd week, the BBB scores of the treated groups (SF/NGF and SF/Alg/NGF) were significantly higher than the SF group. Four and eight weeks post-transplantation, the most significant functional recovery was observed in the SF/Alg/NGF group compared with the SF group (p b 0.05). Taken together, these findings implied that SF/Alg enhance the therapeutic effects of NGF on functional improvement of locomotor activity after SCI.
The development of a safe and efficient treatment for spinal cord injuries is greatly complicated by the existence of a highly complex injury environment [31]. While the axons of injured spinal cord have regenerative potential, they are hindered by various pathophysiological changes and complications following an injury [32]. As a remedy to overcome this hurdle, neural tissue engineering has received considerable attention in recent years. A biomaterial scaffold synthesized from either a natural or synthetic polymer can help prevent the formation of scar tissue and concentrate neurotrophic growth factors while promoting axonal regeneration between the two ends of the injured neural tissue [33–35]. An attractive strategy for repairing injured spinal cord is to incorporate multiple neurotrophic factors in biodegradable and biocompatible microspheres that allow controlled, sustained and localized delivery of
Fig. 7. HE staining results of all experimental groups at 4 W and 8 W after treatment.
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Fig. 8. Immunofluorescence images of NeuN from all experimental groups at 4 W and 8 W after treatment. (Green: NeuN, Blue: DAPI). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
those factors [36]. The Alg is a suitable biomaterial construct providing a cellular mechanical framework of polysaccharide chains that gels by ionic cross linking after mixing aqueous alginate solution with divalent cations such as Ca2+ [20,37]. Alginate hydrogel has been widely used for drug or neurotrophic factors as an injectable vehicle capable of filling cavities in the injured spinal cord, and of providing the substrate for axon attachment and re-growth [23,38]. As shown in Fig. 1, the SEM analysis of Alg indicates a spherical morphology with an average diameter of 198 μm and a smooth surface. Moreover, the water absorption profile of Alg can be used to evaluate the merits of specific Alg as drug carriers. The swelling ratio of the Alg microspheres used in this study was 7.8 ± 0.8, which was similar to values reported previously [25]. Hence, the morphology and high water absorption capability of Alg microspheres suggest that they can be easily loaded with drug. The SF/Alg/NGF composite scaffolds were composed of the following three types of compound: SF as the scaffold material, NGF as the neurotrophic factor, and Alg as the carrier for the NGF. In the present study, the Alg microsphere systems are the ideal carriers for controlled delivery applications because of their ability to encapsulate NGF, as well as
the ease of processing, mechanical properties, biocompatibility, high bioavailability, controlled release rates, stability, and suitability for targeted/localized delivery of different agents. Furthermore, SF scaffold were applied to bridge the gap of the lesion in the spinal cord as contact guidance for axonal growth and to act as a medium to enrich NGF to repair the injured site. The SF/Alg/NGF composite scaffolds exhibited a uniform pore distribution with an average size of 30–45 μm and a porosity of 83.8 ± 0.9% (Fig. 1). The use of SF as a scaffold material not only improved cell proliferation and differentiation but also adherence and migration [8,9,39]. As shown in Fig. 4, SF/Alg/NGF scaffolds exhibited the lowest NGF release rate, which was attributed to the distribution and integration of SF/Alg/NGF throughout the SF scaffold structure. As the release profiles show, the release rate of SF/Alg/NGF was slower than the SF/NGF scaffolds. In SF/NGF scaffolds, most of the NGF was located around the scaffold; thus, NGF was released rapidly from the SF/NGF scaffolds by diffusion. These revealed that most of the drug absorbed by Alg was presented in the particle interior. Besides, the SF/Alg/NGF scaffold had the best sustained-release function and that the Alg were the key
Fig. 9. Immunofluorescence images of MAP2 from all experimental groups at 4 W and 8 W after treatment. (Red: MAP2, Blue: DAPI). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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determining factor in the controlled release of NGF. Besides, in the BBB test, the repair effects of SCI in SF/Alg/NGF group were more significant compared with the SF/Alg group. The roles of NGF as the neurotrophic factor promoting axon regeneration and enhancing functional recovery were well played out. The expression of the neuronal specific marker including MAP2 and NeuN were detected using immunofluorescence method to indicate the situation of nerve repair in the spinal cord after administration. From the data of the immunofluorescence (Figs. 8–9), more MAP2- and NeuN-positive neurons were observed in the spinal cord of the SF/Alg/ NGF group at 4 W after treatment compared with the SF group; and at 8 W, the SF/Alg/NGF group showed more significant effects. These results demonstrated that SF/Alg/NGF for the treatment of rat SCI significantly improved the neurological function of the damaged spinal cord at 4 W following treatment. In addition, at 8 W after treatment, further improvement was observed. To sum up, the Alg were the key determining factor in the controlled release of NGF, and the use of SF as a scaffold material not only improved cell adherence and migration but also proliferation and differentiation. NGF were loaded on the SF/Alg composite scaffolds builds a good micro-environment for growth and repair of the neurons. The underlying mechanism is still ongoing studied in the further research. 5. Conclusion In this study, NGF was loaded on the SF/Alg composite scaffolds to promote neurons outgrowth and regeneration in the SCI rat's model. After eight weeks' treatment, the motor function of rat's from the SF/ Alg/NGF group was improved compared with the SF group. NGF released from SF/Alg composite scaffolds to the central lesion site of SCI significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons. This optimal multi-disciplinary approach of combining biomaterials, controlled-release microspheres and neurotrophic factors offers a promising treatment for the injured spinal cord. Acknowledgments This study was supported financially by the Scientific Research Cultivation and Innovation Fund (21615468), and the Administration of Traditional Chinese Medicine of Guangdong Province (20141067), the High Level University Construction Funds (88016013032), the Natural Science Foundation of China (31271019), and the Science and Technology Project of Guangdong (2011B031300006, 2016ZC0050). References [1] C. National Spinal Cord Injury Statistical, J. Spinal Cord Med. 37 (2014) 355–356. [2] E.Y. Snyder, Y.D. Teng, N. Engl. J. Med. 366 (2012) 1940–1942. [3] J.W. Rowland, G.W. Hawryluk, B. Kwon, M.G. Fehlings, Neurosurg. Focus. 25 (2008) E2.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A bridging SF/Alg composite scaffold loaded NGF for spinal cord injury repair Genlong Jiao a,1, Yongqin Pan b,1, Cunchuang Wang b,⁎, ZhaoXia Li c,⁎, Zhizhong Li a, Rui Guo d,e,⁎⁎ a
Department of Orthopedic, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China Department of General Surgery, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China Department of Rheumatology, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China d Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China e Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China b c
a r t i c l e
i n f o
Article history: Received 22 June 2016 Received in revised form 19 December 2016 Accepted 21 February 2017 Available online 22 February 2017 Keywords: Silk fibroin Scaffold Alginate microspheres Nerve growth factor Spinal cord injury
a b s t r a c t Neurons loss and axons degeneration after spinal cord injury (SCI) gradually give rise to result in functional motor and sensory impairment. A bridging biomaterial scaffold that allows the axons to grow through has been investigated for the repair of injured spinal cord. In this study, we introduced a silk fibroin (SF)-based neurobridge as scaffold enriched with/without nerve growth factor (NGF) that can be utilized as a therapeutic approach for spinal cord repair. NGF released from alginate (Alg) microspheres on SF scaffold (SF/Alg composites scaffolds) to the central lesion site of SCI significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons. This optimal multi-disciplinary approach of combining biomaterials, controlled-release microspheres and neurotrophic factors offers a promising treatment for the injured spinal cord. © 2017 Published by Elsevier B.V.
1. Introduction Spinal cord injury (SCI) is one kind of serious trauma which could cause loss of neurons and degeneration of axons, and further gradually result in the loss of sensory and motor functions [1]. Because of the severity of SCI, there is no effective treatment to completely cure at present. Incidence of SCI presents an increasing tendency, so the discovery of new therapeutic strategies has become a clinically urgent need nowadays. Current treatment to SCI includes surgery to decompress and stabilize the injury, prevention of secondary complications, management of any that do occur, and rehabilitation [1,2]. However, the microenvironment at the spinal cord injury site is rather complicated, and more than one process needs to be regulated in order for axonal regrowth to occur [3]. Not only should hindering factors, such as gliosis or inflammation, be minimized, but the controlled release of necessary neurotrophic factors should be
⁎ Corresponding authors. ⁎⁎ Correspondence to: R. Guo, Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China. E-mail addresses: [email protected] (C. Wang), [email protected] (Z. Li), [email protected] (R. Guo). 1 The authors contributed equally to this work.
sustained; so the theoretical approach to repairing an injured spinal cord is to regenerate damaged axons through the site of injury [4]. A bridging biomaterial construct that allows the axons to grow through has been investigated for the repair of injured spinal cord [5]. Due to the hostility of the microenvironment in the lesion, multiple conditions need to be fulfilled to achieve improved functional recovery. An attractive strategy for repairing injured spinal cord is to design of a new bioactive scaffold that can bridge the gap of the lesion as contact guidance for axonal growth and act as a vehicle to deliver neurotrophic factors in order to modify the microenvironment [6,7]. Silk fibroin (SF), a non-toxic, non-immunogenic structural protein [8], has been successfully explored for the nerve repair research [9]. Scaffolds fabricated from SF delivering neurotrophin (NT)-3 were applied in the treatment of a rat SCI model, and the study showed improved axonal growth [10]. In another study, bi-layer SF scaffolds and small intestinal submucosa matrices could support bladder tissue regeneration in SCI rat's model [11]. Nerve growth factor (NGF) is an important member of the neurotrophin family and an important regulator of neural survival, development, function, and plasticity [12]. It has been indicated that NGF shows neuroprotective effects and improves the recovery of SCI [13–15]. NGF was selected due to its stable and high binding properties, as well as the regulation of neural survival, development, function, and plasticity. Although NGF is important for their mitotic and differential properties for endogenous neural progenitors and their ability to
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accelerate neovascularisation, the application of NGF was limited in the clinic because of its short half-life, rapid dilution in the body [16]. So it is important to localize and sustain release of NGF to the nerve injury site. Alginate (Alg) is one of the most commonly used natural biopolymer, its biodegradability and drug release properties could be regulated by composition, sequence, G-block length, and molecular weight [17–19]. At present, biodegradable Alg microspheres were widely applied in the drug controlled release system [20–22]. It has been reported that Alg microspheres were used as an injectable vehicle capable of filling cavities in the SCI, and of providing the substrate for axon attachment and re-growth [23,24]. In order to overcome the technical bottlenecks in the treatment of SCI, we designed a promising therapeutic strategy combining biomaterials, controlled-release microspheres and neurotrophic factors for the injured spinal cord: SF-based neurobridge as the scaffold and Alg-microsphere as the controlled-release delivery carrier. NGF were loaded on the SF/Alg composite scaffolds to promote neurons outgrowth and regeneration in the SCI rats model, and the combined therapeutic effects of SF/Alg/NGF composite scaffold for SCI repair were evaluated.
2. Materials and methods
other chemicals were of analytical reagent quality and used without further processing. 2.2. Preparation of the SF/Alg/NGF composite scaffolds NGF-loaded Alg microspheres were prepared according to the literature [17,25,26]. Briefly, sodium Alg was dissolved in purified water to a concentration of 2.0% (w/v) with overnight stirring at room temperature. NGF (100 μg/mL), and then added to the Alg solution by shaking gently for 10 min. The NGF-loaded Alg solution was then injected into a 50 mM CaCl 2 solution to form microspheres via a syringe and a high direct-current (DC) voltage. The microspheres were hardened for 15 min and then washed for three cycles using purified water. The final concentration of the SF solution was ~ 4% (w/v), which was determined gravimetrically after freeze-drying for 24 h to remove any remaining solvent. The SF/Alg/NGF scaffold was produced by mixing 1 mL of 4% (w/v) SF solution and 5 mg of Alg/NGF, and then pouring the solution (100 μL) into a 96-well plate and freeze-drying for 24 h. The NGF/ SF scaffold was fabricated by mixing 1 mL of 4% (w/v) SF solution and 4 μL of NGF solution (0.5 μg/μL) and then pouring the mixture (100 μL) into a 96-well and freeze-drying for 24 h. Pure SF scaffolds were also prepared.
2.1. Materials 2.3. Scanning electron microscopy Calcium alginate and pentobarbital was obtained from Sigma (USA). B. mori silkworm cocoons used in the experiment were kindly donated by Sijia Min from Zhejiang University. NGF was purchased from R&D Systems (USA). Dulbecco's-modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). PC12 cells were donated by the Bio-Engineering Institute of the Jinan University. Cell Counting Kit-8 (CCK-8, Beyotime, China). All
Scaffolds of the various SF/Alg/NGF blends were cut out and inspected before and after methanol treatment under a stereomicroscope (Leica Microsystems, Switzerland). Methanol treated samples were also observed by scanning electron microscopy (SEM). SEM samples were coated with platinum prior to evaluation with a LEO 1530 GEMINI scanning electron microscope (Zeiss, Cambridge, UK).
Fig. 1. SEM images of Alg microsphere (A), SF scaffolds (B) and SF/Alg scaffolds (C).
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intervals, 1 mL supernatant was removed for analysis and replenished with 1 mL fresh buffer for continuing incubation. The concentration of NGF released from the scaffolds was quantified by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's guidelines (R&D Systems, USA). All release experiments were carried out in triplicate.
2.6. Bioactivity of the released NGF
Fig. 2. FTIR spectra of Alg, SF and SF/Alg.
Histological sections (see below) were inspected by light microscopy (IX71, Olympus, Japan). 2.4. Structure analysis The infrared (IR) spectra of SF, Alg and SF/Alg were obtained using a Fourier transform infrared (FTIR) spectrometer (Vertex 70; Bruker, Billerica, MA). The IR spectra in the absorbance mode were recorded using a diamond crystal plate and obtained in the spectral region 400–4000 cm − 1 with a resolution of 4 cm − 1 and 20 scans per sample. Raman spectra were recorded on a Bruker MultiRam FT-Raman spectrometer equipped with a cooled Ge-diode detector. The excitation source was a Nd3t-YAG laser (785 nm) in the backscattering (180°) configuration. The focused laser beam diameter was about 100 mm, the spectral resolution 4 cm− 1, and the laser power at the sample about 100 mW. 2.5. In vitro release of NGF The in vitro release of SF/NGF and SF/Alg/NGF scaffolds was evaluated in samples immersed in 50 mL phosphate-buffered saline (PBS), pH 7.4, at 37 °C with shaking at 100 rpm. At predetermined time
The bioactivity of NGF released from SF/NGF and SF/Alg/NGF samples in vitro was examined by determining its ability to stimulate the proliferation of PC12 cells. Released NGF samples collected at day 1, 7 and 14 from SF/NGF and SF/Alg/NGF in 1 mL medium (same with the cell culture medium) were used. PC12 cells were cultured in DMEM supplemented with heat-inactivated 10% FBS, 5% horse serum, and antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin) and incubated in a humidified incubator at 37 °C with 5% CO2. Cells were seeded on 96-well plates (4000 cells/well) at 37 °C, 5% CO2. After cell attachment (24 h), the culture medium was then discarded and an additional 100 μL/well fresh complete DMEM was added. After incubation for another 24 h, 10 μL of the collected released NGF samples from SF/NGF and SF/Alg/NGF medium at different sampling times (at day 1, 7 and 14) with the same NGF concentration of 1.5 μg/mL adjusted by fresh medium, was added to each well, respectively. Positive control condition was also studied. For the positive control condition, 10 μL of fresh release medium with 15 ng standard NGF was added. After 48 h incubation, cell viability was determined using a CCK-8 assay (Beyotime, China) with a fluorescence plate reader, according to the manufacturer's instructions.
2.7. Animals 72 healthy adult female Wistar rats (240–260 g) were obtained from the Experimental Animal Center of Guangdong (Guangzhou, China). The rats were housed in a specific pathogen-free room at a constant temperature of 25 °C and humidity of 45%. The present study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Science (CAMS, China).
Fig. 3. Raman spectra of SF and SF/Alg.
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2.8. Animal model establishment, grouping and implantation treatment The SCI SF was established in accordance with the modified Allen's method. The rats were anesthetized with 400 mg/kg chloral hydrate via intraperitoneal injection. A laminectomy was subsequently performed on the entire spinous process, the vertebral plate of T9 and part of the vertebral plate of T8 and T10, to expose the dorsal (posterior) surface of the spinal cord. The exposed spinal cord at the T9 level was vulnerated with a 10 g weight dropped from a height of 2.5 cm (vulnerating energy, 25 g/cm). Following injury, the rats were randomly divided into three groups: the SF group (n = 24), SF/NGF group (n = 24), SF/Alg/NGF group (n = 24). The prepared scaffolds (SF, SF/NGF and SF/Alg/NGF) were implanted into the wounded site of the rats with SCI respectively. 2.9. Behavior and histological detection At week two, four, six and eight following transplantation, an inclined plane test was conducted and Basso, Beattie, Bresnahan (BBB) locomotor rating scale values were obtained for the rats in the control and transplantation groups. Samples collected from the rats at week four and eight were stained with hematoxylin-eosin (HE) dyes, to examine the histological changes. 2.10. Immunofluorescence Serial 5-μm paraffin-embedded sections of rats' spinal cord were prepared to detect the protein expression of MAP2 and NeuN in each experimental group. After dewaxing, sections were incubated in 3% H2O2 for 15 min and then in blocking solution for 1 h at 37 °C. Subsequently, the sections were incubated at 4 °C overnight with the following primary antibodies: anti-MAP2 (Sigma, 1:500), anti-NeuN (Abcam, 1:500). After washed three times with PBS, sections were incubated with DyLight® secondary antibodies (Abcam, 1:1000) for 2 h at 37 °C. The results were photographed through a light microscope (IX71, Olympus, Japan), and micrographs were analyzed using Image-Pro plus 6.0 software. 2.11. Statistical analysis All experimental data were analyzed with the repeated one-way analysis of variance (ANOVA), which was followed by a Newman– Keuls post hoc test for multiple comparisons using SPSS 17.0 software (SPSS Inc., USA). p b 0.05 was considered to be statistically significant.
Fig. 4. In vitro NGF release profiles from SF/NGF and SF/Alg/NGF scaffolds.
3.2. FTIR and Raman analysis The infrared (IR) spectra of SF, Alg and SF/Alg were performed and shown in Fig. 2, the presence of SF could be observed in SF/Alg composite; characteristic SF peaks at 676, 1064, 1528 and 1668 cm−1 were observed [27]. The Raman spectrum (Fig. 3) of SF showed amide I band at 1668–1640 cm−1 and a complex amide III band with components at 1272 and 1253–1245 cm−1 [28]. In the Raman spectra of the SF scaffolds some bands are definitely ascribable to side-chain vibrations of specific amino acids. Among them, the Raman bands of Tyr at 1615, 1211, 1174, 854, 830, and 644 cm−1 appeared with higher intensity in SF. The bands of SF (1668, 1335 and 854 cm−1) were observed in SF/ Alg scaffolds.
3.3. In vitro NGF release NGF release studies were carried out to evaluate the ability of Alg to control the release of the loaded drug. As shown in Fig. 4, in SF/NGF scaffolds, N65% of the NGF was rapidly released within 4 h. After 72 h, almost all NGF loaded in the SF/NGF scaffold was released. The drug release rate was the slowest in the SF/Alg/NGF scaffolds; NGF was released gradually from the SF/Alg/NGF scaffolds over 240 h, only about 8.23% of the NGF released in the first 4 h period.
3. Results 3.1. Characterisation of Alg microspheres and composite scaffolds From the SEM micrographs of Fig. 1A, all the Alg microspheres had a spherical shape and that their surfaces were pitted with holes. The mean dry diameter of the microsphere was 198 ± 5.9 μm. The dry microspheres tended to swell when immersed in water and became transparent; the average diameter microsphere after swelling in water was 401 ± 7.8 μm. The Alg microspheres fabricated using a water-in-oil process had a porous structure, with an average swelling ratio of 7.8 ± 0.8, which suggested that these microspheres could be applied as drug carriers. Fig. 1B shows the SEM images of the SF scaffold, the pore distribution was uniform, with an average pore size of 75–90 μm. The porosity of the SF scaffold was 96.2 ± 0.7%. A typical SEM image of the SF/Alg scaffold is shown in Fig. 1C. Compared with Fig. 1B, the SF/Alg scaffold exhibited an even distribution of pores, with an average pore size of 30–45 μm (porosity: 83.8 ± 0.9%). Alg microspheres were observed in the pore walls of the SF scaffold and were well distributed; holes were also observed in the microsphere surface.
Fig. 5. Biological activities of NGF released from SF/NGF and SF/Alg/NGF scaffolds. (*p b 0.05 vs. SF/NGF).
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3.6. Histological observation Pathological changes of the spinal cord samples from the experimental groups at 4 W and 8 W after treatment were shown in Fig. 7. The neurons in the spinal cord of the SF group were shrunken or had pale homogenous cytoplasm. Obvious improvement on tissue repairment were observed in both SF/NGF and SF/Alg/NGF group at 4 W after treatment compared with the SCI group; and at 8 W, the two groups showed more ameliorative effects with less necrosis and karyopyknosis. These results further strengthened that SF/Alg/NGF administration has a neuroprotective effect and improves the SCI recovery. 3.7. Promoting neurons growth Fig. 6. The BBB locomotion scores of all experimental groups from 2 weeks to 8 weeks after treatment (*p b 0.05 vs. SCI group).
3.4. Bioactivity of released NGF in the cell proliferation assay To examine the biological activity of released NGF, a CCK-8 assay was used to evaluate the PC12 cells proliferation. From the data in Fig. 5, the cell viability of SF/Alg/NGF group at day 1, 7 and 14 showed no significant difference by comparing numbers between the different time points. Moreover, compared with SF/ NGF, SF/Alg/NGF promoted cell proliferation and viability (p b 0.05 vs. SF/NGF), suggesting that Alg incorporation retained the bioactivity of NGF.
Immunofluorescence detection was used to examine the expression of the neuronal specific marker after transplantation. Microtubuleassociated protein 2 (MAP2) is a neuronal protein that regulates the structure and stability of microtubules, neuronal morphogenesis, cytoskeleton dynamics, and organelle trafficking in axons and dendrites [29]. Neuronal nuclei (NeuN) is a nuclear protein expressed in most post-mitotic neurons of the central and peripheral nervous systems [30]. From the results of the immunofluorescence (Figs. 8–9), more MAP2- and NeuN-positive neurons were observed in the spinal cord of the SF/Alg/NGF and SF/Alg/NGF groups at 4 W after treatment compared with the SCI group; and at 8 W, the two groups showed more significant effects. 4. Discussion
3.5. Animal behavior function The open field locomotor function was assessed using the BBB scoring method at 2 W, 4 W, 6 W, 8 W after scaffolds transplantation. As shown in Fig. 6, all the rats displayed complete hind limb paralysis after SCI. In the following 2–8 weeks, locomotor performance substantially improved. Beginning in the 2nd week, the BBB scores of the treated groups (SF/NGF and SF/Alg/NGF) were significantly higher than the SF group. Four and eight weeks post-transplantation, the most significant functional recovery was observed in the SF/Alg/NGF group compared with the SF group (p b 0.05). Taken together, these findings implied that SF/Alg enhance the therapeutic effects of NGF on functional improvement of locomotor activity after SCI.
The development of a safe and efficient treatment for spinal cord injuries is greatly complicated by the existence of a highly complex injury environment [31]. While the axons of injured spinal cord have regenerative potential, they are hindered by various pathophysiological changes and complications following an injury [32]. As a remedy to overcome this hurdle, neural tissue engineering has received considerable attention in recent years. A biomaterial scaffold synthesized from either a natural or synthetic polymer can help prevent the formation of scar tissue and concentrate neurotrophic growth factors while promoting axonal regeneration between the two ends of the injured neural tissue [33–35]. An attractive strategy for repairing injured spinal cord is to incorporate multiple neurotrophic factors in biodegradable and biocompatible microspheres that allow controlled, sustained and localized delivery of
Fig. 7. HE staining results of all experimental groups at 4 W and 8 W after treatment.
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Fig. 8. Immunofluorescence images of NeuN from all experimental groups at 4 W and 8 W after treatment. (Green: NeuN, Blue: DAPI). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
those factors [36]. The Alg is a suitable biomaterial construct providing a cellular mechanical framework of polysaccharide chains that gels by ionic cross linking after mixing aqueous alginate solution with divalent cations such as Ca2+ [20,37]. Alginate hydrogel has been widely used for drug or neurotrophic factors as an injectable vehicle capable of filling cavities in the injured spinal cord, and of providing the substrate for axon attachment and re-growth [23,38]. As shown in Fig. 1, the SEM analysis of Alg indicates a spherical morphology with an average diameter of 198 μm and a smooth surface. Moreover, the water absorption profile of Alg can be used to evaluate the merits of specific Alg as drug carriers. The swelling ratio of the Alg microspheres used in this study was 7.8 ± 0.8, which was similar to values reported previously [25]. Hence, the morphology and high water absorption capability of Alg microspheres suggest that they can be easily loaded with drug. The SF/Alg/NGF composite scaffolds were composed of the following three types of compound: SF as the scaffold material, NGF as the neurotrophic factor, and Alg as the carrier for the NGF. In the present study, the Alg microsphere systems are the ideal carriers for controlled delivery applications because of their ability to encapsulate NGF, as well as
the ease of processing, mechanical properties, biocompatibility, high bioavailability, controlled release rates, stability, and suitability for targeted/localized delivery of different agents. Furthermore, SF scaffold were applied to bridge the gap of the lesion in the spinal cord as contact guidance for axonal growth and to act as a medium to enrich NGF to repair the injured site. The SF/Alg/NGF composite scaffolds exhibited a uniform pore distribution with an average size of 30–45 μm and a porosity of 83.8 ± 0.9% (Fig. 1). The use of SF as a scaffold material not only improved cell proliferation and differentiation but also adherence and migration [8,9,39]. As shown in Fig. 4, SF/Alg/NGF scaffolds exhibited the lowest NGF release rate, which was attributed to the distribution and integration of SF/Alg/NGF throughout the SF scaffold structure. As the release profiles show, the release rate of SF/Alg/NGF was slower than the SF/NGF scaffolds. In SF/NGF scaffolds, most of the NGF was located around the scaffold; thus, NGF was released rapidly from the SF/NGF scaffolds by diffusion. These revealed that most of the drug absorbed by Alg was presented in the particle interior. Besides, the SF/Alg/NGF scaffold had the best sustained-release function and that the Alg were the key
Fig. 9. Immunofluorescence images of MAP2 from all experimental groups at 4 W and 8 W after treatment. (Red: MAP2, Blue: DAPI). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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determining factor in the controlled release of NGF. Besides, in the BBB test, the repair effects of SCI in SF/Alg/NGF group were more significant compared with the SF/Alg group. The roles of NGF as the neurotrophic factor promoting axon regeneration and enhancing functional recovery were well played out. The expression of the neuronal specific marker including MAP2 and NeuN were detected using immunofluorescence method to indicate the situation of nerve repair in the spinal cord after administration. From the data of the immunofluorescence (Figs. 8–9), more MAP2- and NeuN-positive neurons were observed in the spinal cord of the SF/Alg/ NGF group at 4 W after treatment compared with the SF group; and at 8 W, the SF/Alg/NGF group showed more significant effects. These results demonstrated that SF/Alg/NGF for the treatment of rat SCI significantly improved the neurological function of the damaged spinal cord at 4 W following treatment. In addition, at 8 W after treatment, further improvement was observed. To sum up, the Alg were the key determining factor in the controlled release of NGF, and the use of SF as a scaffold material not only improved cell adherence and migration but also proliferation and differentiation. NGF were loaded on the SF/Alg composite scaffolds builds a good micro-environment for growth and repair of the neurons. The underlying mechanism is still ongoing studied in the further research. 5. Conclusion In this study, NGF was loaded on the SF/Alg composite scaffolds to promote neurons outgrowth and regeneration in the SCI rat's model. After eight weeks' treatment, the motor function of rat's from the SF/ Alg/NGF group was improved compared with the SF group. NGF released from SF/Alg composite scaffolds to the central lesion site of SCI significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons. This optimal multi-disciplinary approach of combining biomaterials, controlled-release microspheres and neurotrophic factors offers a promising treatment for the injured spinal cord. Acknowledgments This study was supported financially by the Scientific Research Cultivation and Innovation Fund (21615468), and the Administration of Traditional Chinese Medicine of Guangdong Province (20141067), the High Level University Construction Funds (88016013032), the Natural Science Foundation of China (31271019), and the Science and Technology Project of Guangdong (2011B031300006, 2016ZC0050). References [1] C. National Spinal Cord Injury Statistical, J. Spinal Cord Med. 37 (2014) 355–356. [2] E.Y. Snyder, Y.D. Teng, N. Engl. J. Med. 366 (2012) 1940–1942. [3] J.W. Rowland, G.W. Hawryluk, B. Kwon, M.G. Fehlings, Neurosurg. Focus. 25 (2008) E2.
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