Polycaprolactone Nanofibers Containing Vascular Endothelial Growth

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Polycaprolactone Nanofibers Containing Vascular Endothelial Growth Factor-Encapsulated Gelatin Particles Enhance Mesenchymal Stem Cell Differentiation and Angiogenesis of Endothelial Cells Yong-Chao Jiang, Xiao-Feng Wang, Yi-Yang Xu, Yu-Hui Qiao, Xin Guo, Dong-Fang Wang, Qian Li, and Lih-Sheng Turng Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00870 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 11, 2018

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Polycaprolactone Nanofibers Containing Vascular Endothelial Growth Factor-Encapsulated Gelatin Particles Enhance Mesenchymal Stem Cell Differentiation and Angiogenesis of Endothelial Cells Yong-Chao Jianga,b,c,d, Xiao-Feng Wanga,b, Yi-Yang Xua,c,d, Yu-Hui Qiaoa,b, Xin Guoa,b, Dong-Fang Wanga,b, Qian Lia,*, and Lih-Sheng Turngc,d,* a

National Center for International Research of Micro-Nano Molding Technology, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China

b

School of Mechanics and Engineering Science, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China

c

Department of Mechanical Engineering, University of Wisconsin–Madison, 1513 University Avenue, Madison, WI 53706, USA

d

Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, 1513 University Avenue, Madison, WI 53705, USA

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KEYWORDS

Nanofibers,

growth

factor,

mesenchymal

stem

cells

(MSCs),

endothelial

differentiation, angiogenesis.

ABSTRACT

During the regeneration of tissues and organs, growth factors (GFs) play a vital role by affecting cell behavior. However, because of the low half-life time and quick degradation of GFs, their stimulations on cells are relatively short and discontinuous. In this study, a releasing scaffold platform, consisting of polycaprolactone (PCL) nanofibers and vascular endothelial growth factor (VEGF)-encapsulated gelatin particles, was developed to extend the influence of GFs on mesenchymal stem cells (MSCs) and endothelial cells (ECs). The results showed that this kind of scaffold can direct the differentiation of MSCs to ECs and maintain the stability of the tubular structure, an indicator of the angiogenesis ability of ECs, for an extended period of time.

Therefore,

the

results

suggest

the

potential

application

of

PCL/VEGF-encapsulated gelatin particles (PCL/VGPs) as a growth factor (GF)-releasing scaffold platform in vascular tissue engineering.

INTRODUCTION Tissue engineering scaffolds have great potential in regenerative medicine, which is crucial for clinical therapy.1-2 Synthetic polymers, with superior and tunable physical and chemical properties, have been electrospun to engineer scaffolds for 2 ACS Paragon Plus Environment

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supporting cell growth and guiding cell behavior.3-4 However, current limitations of synthetic polymers, including biocompatability and biofunctionability, are still choke points in the tissue engineering field.5-6 Even though in previous studies electrospun PCL/gelatin scaffolds were developed to enhance cell proliferation and migration on engineered scaffolds,7-8 directional guidance from electrospun scaffolds on the specific differentiation of pluripotent stem cells has not yet been fully explored. The differentiation of stem cells, as well as the regeneration of tissues and organs, is typically driven by the effects of growth factors (GFs).9-11 At present, electrospun fibrous scaffolds functionalized with GFs have been widely utilized as tissue engineering scaffolds to help promote cell repair and tissue regeneration.12-14 Nevertheless, current methods, which are aimed at integrating GFs with scaffolds, such as dip-coating and covalent binding, yield relatively low loading efficiencies and an initial robust release behavior that tapers off.15-16 From the investigation reported by Lee et al., the author found that a coacervate-coated nanofiber with VEGF and transforming growth factor beta-3 (TGF-β3) could improve neovascularization in a mouse skin flap model. However, both of the GFs showed robust release profiles in 2 days.12 Thus, a stable GF-releasing scaffold platform is needed for tissue engineering applications.17-19 The encapsulation of active molecules allows for their protection from degradation in physical and biological fluids, and provides for their sustained and controlled release over space and time.20-21 For instance, to achieve an optimized oral suspension, Ghosal et al. studied the average particle size and drug entrapment 3 ACS Paragon Plus Environment


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efficiency of naringin-loaded PCL microspheres affected by the concentration of PCL, polyvinyl alcohol (PVA), and hydroxypropyl methylcellulose (HPMC), but they failed to include the effects of microspheres on cell behavior.22 Therefore in our study, vascular endothelial growth factor (VEGF)-encapsulated gelatin particles were manufactured and electrospun into polycaprolactone (PCL) nanofibers as scaffolds for cell growth. The properties of scaffolds and VEGF release behaviors were analyzed. Then these scaffolds were employed to regulate human mesenchymal stem cell (MSC) differentiation into endothelial cells (ECs). Moreover, paracrine interactions between scaffolds and ECs were also investigated. MATERIALS AND METHODS Manufacturing of gelatin particles (GPs) and VEGF-encapsulated gelatin particles (VGPs). GPs were produced by a two-step desolvation method reported by Azimi et al.21 Briefly, gelatin (Type B, Sigma–Aldrich) was dissolved in distilled water with gentle stirring at 37 °C. Gelatin with a low molecular weight was discarded with acetone addition, and then the residual gelatin was redissolved with distilled water. For VEGF-encapsulated gelatin particles (VGPs), VEGF was introduced at a weight percentage of 10% to gelatin. Fluorescein isothiocyanate (FITC) was added at a concentration of 2.5 mg/mL to label the gelatin. Acetone was then added dropwise into the solution under gentle stirring at 37 °C to form sub-micron or micron-scale gelatin particles (GPs) and VGPs, and glutaraldehyde was used to crosslink the particles for 30 min. The morphology and diameter of GPs and VGPs were measured by digital LEO GEMINI 1530 SEM (ZEISS) and Zetasizer 4 ACS Paragon Plus Environment

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Nano equipment (ZSP, Malvern). Moreover, the loading efficiency of VEGF within GPs was measured via the BCA Protein Assay Kit (Pierce, USA). Then the GPs and VGPs were freeze-dried separately for future use. Processing of PCL fibrous scaffolds containing GPs and VGPs. The GP- and VGP-loaded PCL nanofibrous scaffolds (denoted as PCL/GPs and PCL/VGPs, respectively) were fabricated via electrospinning as previously described.7 Briefly, a polymer solution with a concentration of 10 wt% was prepared by dissolving PCL and GPs (or VGPs) at a weight ratio of 7:3 in chloroform. Particle distribution within the PCL nanofibers was observed via a confocal laser microscopy (Nikon A1RS, Japan). VEGF release profile from PCL scaffolds. The PCL/VGPs scaffold was suspended in a phosphate buffered solution (PBS) with 1% trypsin and agitated in a 37 °C water bath to test the in vitro VEGF release profile from the nanofibers. At each predetermined time point, 1 ml of supernatant was withdrawn to measure the VEGF amount, and 1 mL of fresh PBS was added back into the culture plate, which was then returned to the shaking bath. Water contact angle (WCA) test of fibrous scaffolds. The surface water contact angles of scaffolds in different groups were tested through the sessile drop method in air at room temperature using a video contact angle instrument (DataphysicsOCA 15, Germany). The drop size was set as 10 µL. Three specimens for each group were tested to calculate the average value. Tensile test of fibrous scaffolds. The tensile behaviors of different scaffolds were tested by an Instron 5967 universal testing machine with a 50 N load cell. The 5 ACS Paragon Plus Environment


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experiments were performed at room temperature (25 °C) and atmospheric conditions (relative humidity of 20 ± 5%), with a crosshead speed of 5 mm/min. The specimens were prepared by cutting strips of fibrous scaffolds 60 mm long by 10 mm wide, whereas the thickness was measured at around 0.3 mm. Each specimen was soaked in PBS for 1 h before being measured. The slope of the linear portion of the stress–strain curve was calculated as the tensile modulus. Five specimens were tested for each ratio and the average values and standard deviations were reported. Thermal properties of fibrous scaffolds. Differential scanning calorimetry (DSC) was used to determine the thermal properties and crystalline behavior of fibrous scaffolds. Samples weighing 6–8 mg were encapsulated in aluminum pans and heated from 20 °C to 180 °C under the protection of a nitrogen atmosphere and an empty pan was used as a control. Particle-loaded scaffolds affect MSC differentiation to ECs. In addition to culturing MSCs on GP- and VGP-loaded PCL fibrous scaffolds, MSCs cultured on PCL scaffolds were treated as negative controls, while MSCs cultured on PCL scaffolds with 50 ng/mL VEGF during differentiation were set as positive controls (denoted as PCL/VEGF).23 Culture medium was changed every two days. After culturing on different scaffolds for 10 days, the total RNA in MSCs was extracted using Trizol (Invitrogen, USA), reverse-transcribed using a high capacity cDNA reverse transcription kit (AB Applied Biosystems, USA) following the manufacturer's instructions, and then amplified using SYBR Green Supermix (Bio-Rad, USA) with primers for detecting NANOG, octamer-binding transcription factor 3/4 (OCT 3/4), 6 ACS Paragon Plus Environment

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platelet endothelial cell adhesion molecule (CD31), von Willebrand factor (vWF), SOX 18, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), and the internal control ubiquitin C (UBC), as listed in Table 1. Apart from the quantitative polymerase chain reaction (qPCR), western blotting (WB) was used to quantify the protein amount of interest in each group. Particle-loaded scaffolds affect EC angiogenesis. In order to study the effect of VEGF released from PCL/VGP scaffolds on EC angiogenesis, ECs were cultured on culture plates pre-coated with matrigel. Then the PCL/VGP scaffolds were placed in the medium. For the angiogenesis study, PCL scaffolds with 50 ng/mL VEGF during cell culture were set as positive controls (denoted as PCL/VEGF). The tubular structure formed by the ECs was captured under an optical microscope after 4, 12, and 24 hours, and the pictures were measured in terms of emerging nodes and tubes using the NIH ImageJ analysis software.24-25 Statistical analysis. Data were presented as the mean ± standard deviation (SD) unless otherwise specified. One-way analysis of variance (ANOVA) was performed to determine statistical significance. A P-value < 0.05 was considered statistically significant unless otherwise noted. RESULTS AND DISCUSSION Electrospun nanofibrous scaffolds for the regeneration of pathological tissues have been widely investigated in the last decade based on their relatively simple fabrication and morphological structure, which is similar to the natural extracellular 7 ACS Paragon Plus Environment


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matrix (ECM).12-13, 26 However, the straightforward use of nanofibers is insufficient to improve their influence on cells and thereby requires further modification with specific biofunctional factors. Likewise, an exogenous GF delivery platform comprised of poly (lactic-co-glycolic acid) (PLGA) nanofibers, coacervate-coated with VEGF and transforming growth factor beta 3 (TGF-β3), was employed to improve neovascularization in skin flaps.12 Although their results showed that this kind of modification could reduce necrosis and cause blood perfusion in skin flap areas 10 days after implantation, the initial burst release of GFs still remained. Considering that nanoparticles have been widely used in biological studies,27-28 and therefore in our study, to extend the release time of GFs to help regenerate the endothelium, gelatin particles as GF-delivery cargos were prepared and used to prevent the quick degradation of VEGF from the electrospun PCL fibers, as shown in Figure 1. According to Figure 2, both GPs (Figure 2A) and VGPs (Figure 2B) showed spherical morphologies and uniform distributions, while VGPs had a larger mean diameter (1102.0 ± 202.6 nm) than GPs (729.1 ± 105.7 nm). According to previous publications, the isoelectric point of gelatin is around 5,29 while VEGF is around 8.5.30 Therefore, during the fabrication of VGPs under physiological conditions, electrostatic interaction between positively charged VEGF molecules and negatively charged gelatin could form particles with a larger mean diameter than GPs. In addition, gelatin is a natural polymer obtained by the partial hydrolysis of collagen,31 so the protein concentration of VGPs was obviously higher than that of GPs (Figure 8 ACS Paragon Plus Environment

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2C). Figure 2D–F showed separate morphologies and fiber mean diameters for PCL, PCL/GPs, and PCL/VGPs. Even though the addition of GPs and VGPs increased the fiber mean diameter, the nanofibrous structure remained and no obvious defects were found across the three groups. The results above confirmed that VEGF was successfully encapsulated in the GPs. Owing to the stretching force during electrospinning,32 an aligned distribution of GPs and VGPs along the axis of the fibers was found in the fluorescence staining images (Figure 3A and B). Traditionally, gelatin will degrade in 10 days in vitro,31 while in our study, the PCL/VGPs scaffold exhibited a more gradual release of VEGF over 10 days because of the crosslinking treatment on the particles during fabrication, suggesting that it could act as a sustaining release platform in tissue engineering (Figure 3C). It could also be found that the release rate in the first 48 h was faster than what followed, which can be attributed to the initial diffusion of gelatin-unbound VEGF and subsequent VEGF released, along with particle degradation. In other words, the first–stage release of VEGF was diffusion-dominated, while the second-stage release was degradation-dominated.29, 31 Apart from that, addition of GPs and VGPs dramatically decreased the water contact angle of the fibrous scaffolds, with 54.1 ± 4.0° and 51.8 ± 3.2° for PCL/GPs and PCL/VGPs, respectively (Figure 3D). The lower WCA was related to a more hydrophilic surface, which is more beneficial for cell attachment and migration on electrospun scaffolds in tissue engineering.7, 33 It was also found that the tensile modulus of PCL/GPs and PCL/VGPs increased almost 1.7 and 2 times, respectively, compared to the PCL 9 ACS Paragon Plus Environment


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group (Figure 3E). During electrospinning, PCL fibers were positively charged according to previous research by Xiao-feng Wang et al.34 Thus, electrostatic interactions occurred between PCL fibers and gelatin particles, which led to an increased tensile modulus. Additionally, the existence of VEGF also affected the tensile modulus due to interactions with GPs. It is worth noting that the elongation and tensile strength of PCL scaffolds weakened after GPs and VGPs addition (Figure 3F and G), however the developed modulus still make these scaffolds suitable for vascular tissue engineering.35 The thermal properties of PCL, PCL/GPs, and PCL/VGPs are included in Figure 3H. Because the electrostatic interactions mentioned above inhibited the movement of molecular chains, the crystallization of PCL decreased with the introduction of GPs and VGPs, from 62.7 J/g in the PCL group to 45.7 J/g in the PCL/VGPs group. Similarly, the degradation temperature of protein increased from 118.7 °C to 129.7 °C, and the heat enthalpy increased from 49.4 J/g to 58.1 J/g in PCL/GPs and PCL/VGPs, respectively. These changes revealed the influence of GPs and VGPs on the mechanical behaviors of fibrous scaffolds. VEGF has been widely reported to guide MSC differentiation to ECs, which are a reliable cell source for successful vascular tissue engineering endeavors.23, 36-37 For instance, Du et al. found that, during endothelial differentiation, MSCs induced by VEGF showed EC phenotypes in culture.38 Therefore in our study, the effects of PCL/GPs and PCL/VGPs on MSCs were investigated via qPCR and WB in order to verify their biofunctionability. Based on our previous study,7 the addition of GPs increased the cell numbers by providing more biofunctional anchor points for cell 10 ACS Paragon Plus Environment

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proliferation,39 thus resulting in a higher NANOG expression compared to pure PCL scaffolds (Figure 4A). On the contrary, PCL/VGPs significantly downregulated NANOG and OCT3/4 expression in MSCs, while upregulating CD31 and vWF levels (Figure 4A), indicating the dominating effect of growth factor on MSC differentiation to ECs.40 However, the regulation of several other EC-related genes was not as strong as those in the PCL/VEGF group (Figure 4B), which was attributed to the lower density of VEGF released from PCL nanofibers. The same trend was also found in the WB results (Figure 4C), with more CD31 and vWF protein production in the PCL/VEGF group than in the PCL/VGPs group, and no related signals were found in the PCL and PCL/GPs groups. In addition to MSCs, effects of PCL/VGPs on ECs at different time points were further investigated. As shown in Figures 5 and 6, PCL/VEGF formed denser tubular structures than PCL/VGPs after 4 h, with an almost two-fold increase in nodes and tubes. However, tubular structures in the PCL/VEGF group began to disintegrate after 12 h and eventually disappeared after 24 h, while in the PCL/VGPs group, the average number of nodes and tubes remained around 54.7% and 50.3% of the original after 24 h. Since this kind of structure could reflect the angiogenesis ability of ECs41, the results above indicate that PCL/VGPs could potentially serve as tissue engineering scaffolds to help repair tissues and maintain angiogenesis for long periods of time. CONCLUSION Functional scaffolds are a prerequisite for guiding cell behavior to accomplish tissue repair and regeneration. In our investigation, electrospun PCL nanofibers and 11 ACS Paragon Plus Environment


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gelatin particle-encapsulated VEGFs were integrated to produce growth factor (GF)-releasing scaffolds that could induce MSC differentiation to ECs while stimulating and further stabilizing the tubular structure of ECs. Thus, our approach for GF delivery has potential applications both in stem cell therapies and vascular tissue engineering.

AUTHOR INFORMATION Corresponding Author *Qian Li, E-mail: [email protected]

*Lih-sheng Turng, E-mail: [email protected]

Author Contributions Yon-chao Jiang and Xiao-feng Wang contributed equally to this work. All authors have given approval to the final version of the manuscript.

Funding Sources This work was supported by the International Science & Technology Cooperation Program of China [2015DFA30550] and the National Science Foundation of China [11372286]. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS The authors would like to acknowledge the support of the Wisconsin Institute for Discovery, as well as the technical guidance and support of Professor Wan-Ju Li at the Musculoskeletal Biology and Regenerative Medicine Laboratory at the University of Wisconsin–Madison.

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(36) Ikhapoh, I. A.; Pelham, C. J.; Agrawal, D. K. Synergistic Effect of Angiotensin Ii on Vascular Endothelial Growth Factor-a-Mediated Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells. Stem Cell Res. Ther. 2015, 6, 4-17.

(37) Kook, Y.-M.; Kim, H.; Kim, S.; Lee, K.; Heo, C. Y.; Park, M. H.; Koh, W.-G. Promotion of Vascular Morphogenesis of Endothelial Cells Co-Cultured with Human Adipose-Derived

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Nanofibrous Scaffolds. Nanomaterials 2018, 8 (2) 117-133.

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Table 1. Primer sequences for qPCR analysis. Gene Name UBC

Accession Number NM_021009.4

Primer Sequences (5'-3') F: TGA AGA CAC TCA CTG GCA AGA CCA R: CAG CTG CTT TCC GGC AAA GAT CAA

NANOG

NM_021865.2

F: GCT GAG ATG CCT CAC ACG GAG R: TCT GTT TCT TGA CCG GGA CCT TGT C

OCT3/4

NM_002701.4

F: TGG AGA AGG AGA AGC TGG AGC AAA A R: GGC AGA TGG TCG TTT GGC TGA ATA

vWF

NM_000552.4

F: GAA ATG TGT CAG GAG CGA TG R: ATC CAG GAG CTG TCC CTC A

CD31

NM_000442.4

F: GCA ACA CAG TCC AGA TAG TCG T R: GAC CTC AAA CTG GGC ATC AT

SOX18

NM_018419

F: CTC GCT GGC CTG TAC TAC G R: GTA CTG GTC GAA CTC GGT GA

VEGFR1

NM_002019

F: GCA AAG CCA CCA ACC AGA AG R: ACG TTC AGA TGG TGG CCA AT

VEGFR2

NM_002253

F: CGG TCA ACA AAG TCG GGA GA R: CAG TGC ACC ACA AAG ACA CG

Forward and reverse primers are indicated as "F" and "R", respectively.


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Figure 1. Schematic diagram of assembling VEGF-encapsulated gelatin particles in electrospun PCL fibers. 128x96mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2. SEM images of (A) GPs and (B) VGPs. Inserted images are diameter measurements of GPs and VGPs by ZSP. (C) Protein concentration of GPs before and after VEGF loading. SEM images and fiber mean diameters of (D) PCL, (E) PCL/GPs, and (F) PCL/VGPs scaffolds; scale bar for D–F is 5 µm. 87x45mm (300 x 300 DPI)


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Figure 3. (A–B) Fluorescence staining images of GP and VGP locations within PCL nanofibers. (C) VEGF release profile with respect to time from PCL nanofibers. (D) Water contact angle, (E) tensile modulus, (F) elongation, and (G) tensile strength of different scaffolds. (H) Thermal properties of different scaffolds. 154x141mm (300 x 300 DPI)



Figure 4. (A) Gene expressions of interest from differentiated MSCs cultured on various scaffolds. (B) Heat map comparasion of seven transcripts analyzed by qPCR. (C) Expression levels of EC-associated proteins from differentiated MSCs cultured on various scaffolds 174x177mm (300 x 300 DPI)


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Figure 5. PCL/VGPs and PCL/VEGF scaffolds stimulated EC angiogenesis after 4, 12, and 24 h. 96x54mm (300 x 300 DPI)



Figure 6. Measurement of the average number of (A) nodes and (B) tubes from tubular structures affected by PCL/VGPs and PCL/VEGF. 76x34mm (300 x 300 DPI)


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For Table of Contents Only A releasing scaffold platform, consisting of polycaprolactone (PCL) nanofibers and vascular endothelial growth factor-encapsulated gelatin particles (VGPs), could induce endothelial differentiation of mesenchymal stem cells (MSCs) and maintain the tubular structure of Endothelial cells (ECs). 71x35mm (300 x 300 DPI)


Polycaprolactone Nanofibers Containing Vascular Endothelial Growth

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