Cerium Oxide Nanoparticle Modified Scaffold ... - ACS Publications

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Cerium oxide nanoparticle modified scaffold interface enhances vascularization of bone grafts by activating calcium channel of mesenchymal stem cells Junyu Xiang, Jianmei Li, Jian He, Xiangyu Tang, Ce Dou, Zhen Cao, Bo Yu, Chunrong Zhao, Fei Kang, Lu Yang, Shiwu Dong, and Xiaochao Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00158 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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ACS Applied Materials & Interfaces

Cerium Oxide Nanoparticle Modified Scaffold Interface Enhances Vascularization of Bone Grafts by Activating Calcium Channel of Mesenchymal Stem Cells

Junyu Xiang a, Jianmei Li a, Jian He b, Xiangyu Tang a, Ce Dou a, Zhen Cao a, Bo Yu c, Chunrong Zhao a, Fei Kang a, Lu Yang a, Shiwu Dong a,d,* and Xiaochao Yang a,*

a

Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military

Medical University, Chongqing 400038, China. b

Department of Chemistry, College of Pharmacy, Third Military Medical University, Chongqing

400038, China. c

Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing

400038, China. d

China Orthopedic Regenerative Medicine Group, China.

*Email: [email protected] (S. Dong), [email protected] (X. Yang).

ABSTRACT Insufficient blood perfusion is one of the critical problems that hamper the clinical application of tissue engineering bone (TEB). Current methods for improving blood vessel distribution in TEB mainly rely on delivering exogenous angiogenic factors to promote the proliferation, migration, differentiation and vessel formation of endothelial cells (ECs) and/or endothelial progenitor cells (EPCs). However, obstacles including limited activity preservation, difficulty in controlled release

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and high cost obstructed the practical application of this strategy. In this study, TEB scaffold were modified with cerium oxide nanoparticles (CNPs) and the effects of CNPs existed at the scaffold surface on the growth and paracrine behavior of MSCs were investigated. The CNPs could improve the proliferation and inhibit the apoptosis of MSCs. Meanwhile, the interaction between the cell membrane and the nanoparticle surface could activate the calcium channel of MSCs leading to the raise of intracellular free Ca2+ level, which subsequently augment the stability of HIF-1α. These chain reactions finally resulted in high expression of angiogenic factor VEGF. The improved paracrine of VEGF could thereby promote the proliferation, differentiation and tube formation ability of EPCs. Most importantly, in vivo ectopic bone formation experiment demonstrated this method could significantly improve the blood vessel distribution inside of TEB. Keywords: cerium oxide nanoparticles, mesenchymal stem cells, angiogenesis, vasculogenesis, tissue engineering bone

1. Introduction The treatment of large non-healing bone defect using TEB has been intensively studied over the past decades in aims to satisfy the gigantic clinical requirement.1,2 Bone is highly vascularized tissue as about 10% of resting cardiac output is supplied to the skeleton system.3,4 Despite great advances in bone regeneration based on tissue engineering technologies, the clinical application of TEB is still challenged by tissue necrosis and fibrosis due to the insufficient blood vessel distribution and blood perfusion in the central part of the graft.5-8 The postnatal new blood vessel formation could be achieved by two ways. One is angiogenesis where the new blood vessels are sprouted from the existing vasculature through the recruitment of ECs.9 Another one is vasculogenesis where the EPCs

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differentiate into mature ECs and form new blood vessels by themselves without the support of the existing vasculature.10 In both angiogenesis and vasculogenesis pathways, angiogenic factors play a vital role in initiating, branching, maturating and ending the blood vessel development process.11,12 Within all of the angiogenic factors, VEGF is of great interest due to its special capability to control the new blood vessel formation process via VEGFR2-mediated signaling pathways.13-15 For example, in angiogenesis process, VEGF participates in sending hypoxic signal, increasing the permeability of the endothelial cell layer, ensuring the migration of ECs, and promoting vessel branching.9 In vasculogenesis process, VEGF is indispensable for recruitment, retention, proliferation, migration and differentiation of EPCs.16-18 Theoretically, TEB could be vascularized by either angiogenesis or vasculogenesis. Therefore, precisely control the origin, concentration, and validity of angiogenic factors is crucial for fabrication of a thoroughly vascularized TEB. As for large-sized TEB, the host secreted angiogenic factors are far from sufficient to induce the migration of ECs and/or EPCs into the central part of the graft.19 Although the delivery of exogenous angiogenic factors is thought to be a compensative way, the short activity preservation duration of the delivered factors could compromise the feasibility of this strategy since the development of new blood vessels usually takes several weeks.20 Other obstacles including difficult for controlled release and high cost also hamper the practical application of growth factors delivery strategy. In consideration of these limits, it is necessary to explore new strategies to supply growth factors for TEB vascularization. The in vivo development of bone and blood vessel is intricately connected and mutual promotion to each other.21 This principle has led to the construct of vascularized TEB by co-seeding ECs or EPCs with bone formation cells.22-24 Though the interactions between ECs or EPCs with 3



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bone-forming cells are so complicated and are far from clearly confirmed by now, the utilization of autocrine or paracrine growth factors to mediate the communication between these cells is well known.21,25 As a typical example, MSCs that could secrete more than twenty angiogenic factors have been used as bone-forming cells to promote the proliferation and migration of ECs or EPCs by using the secreted factors.26 Though this clue has been revealed for long time, less studies could turn it into practical vascularized TEB construction. One of the main reasons could be the limited secretion of angiogenic factors that is insufficient for blood vessel growth, especially at the central part of the graft. In view of this hypothesis, the stimulation of bone-forming cells to augment the secretion of angiogenic factors could be a potential way to promote the blood vessel development in TEB. Recently, studies have reported that CNPs have the ability to imitate multiple enzyme properties including superoxide oxidase,27 catalase,28 oxidase29 and phosphatase30. Because most of these enzymes are crucial for controlling the oxygen and reactive oxygen species level in the cells, CNPs have found various biomedical applications such as anti-radiation, anti-inflammatory, ischemic stroke protection, and treatment of dermal wounds.31,32 It is worth noting that CNPs could control the growth,33 differentiation and migration of MSCs.34 Besides, CNPs could also promote the angiogenesis through the modulation of oxygen in ECs.35 Nevertheless, no previous attention has been paid to the effects of CNPs on the paracrine of angiogenic factors by bone-forming cells. In the present study, the effects of CNPs embedded at the TEB scaffold surface on the paracrine of angiogenic factors from MSCs were explored. The promotion of EPCs proliferation, differentiation, tube formation and in vivo new blood vessel formation by utilizing the MSCs secreted angiogenic factor were investigated. The mechanisms of how the CNPs existed at the scaffold and cell interface manipulate the growth and paracrine behaviors of MSCs were also discussed. 4


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2. Materials & methods 2.1. Synthesis of CNPs CNPs with size ~5nm were synthesized by thermal decomposition method using cerium nitrate as precursor according to previous report.36 In brief, 1.0 mmol cerium nitrate was dissolved in 1 mL oleylamine and 5 mL 1-octadecene. The mixture was heated to 80 °C for 30 min and then heated to 260 °C for 2 h. After cooling to room temperature, the nanoparticles were washed with acetone for several times. Finally, the nanoparticles were dispersed in dichloromethane. 2.2. Fabrication of scaffold and CNPs modified scaffold The scaffold without CNPs modification was prepared by immersing cancellous bone in PLLA dichloromethane solution overnight. The PLLA concentrations range from 0.1% to 2.0% (w/v). The CNPs embedded scaffold (termed as scaffold@CNPs in all of the following text) was prepared by immersing cancellous bone in CH2Cl2 solution containing PLLA and CNPs. In detail, PLLA was dissolved in CH2Cl2 with concentration of 2.0% (w/v). The CNPs were mixed with the PLLA solution with CNPs/PLLA ratio (wt%) up to 10%. The cancellous bone was immersed in the mixture overnight. The prepared scaffold and scaffold@CNPs were thoroughly washed by 75% ethanol and immersed in PBS for 24h. The scaffolds were sterilized by epoxyethane before seeding cells. 2.3. Cell culture The mesenchymal stem cell line C3H/10T1/2 was purchased from ATCC. MSCs were cultured in a humidified atmosphere (5% CO2) at 37 ºC, and grown in DMEM/F12 medium supplemented with 10% FBS. The EPCs were separated from the spleen mononuclear cells of C57BL/6 mice by density gradient centrifugation. Mice were sacrificed by cervical dislocation. Spleen was collected 5



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and cut to single cells with scissors. Cell suspension was screened on a 200 mesh sieve and separated by centrifugation at centrifugal force of 400 g for 20 min using Histopaque®-1077 Hybri-Max™ (Sigma) as density medium. After centrifugation, the cells were separated into three layers. The middle layer containing mononuclear cells were collected and washed twice by PBS. The cells were cultured in a humidified atmosphere (5% CO2) at 37 ºC, and grown in DMEM/F12 medium supplemented with 20% FBS. 2.4. Alkaline Phosphatase (ALP) Assay MSCs (1×104) were seeded on scaffold and scaffold@CNPs in 24 plate well. The ALP activity and total protein were detected at 1, 7 and 14 days using Alkaline Phosphatase Assay Kit (Beyotime biotechnology, China) and Bicinchoninic Acid Protein Assay Kit (Beyotime biotechnology, China) according to the manufacturer’s instruction. The values of ALP activity were normalized with respect to the total protein content. 2.5. MSCs cell cycle detection MSCs (1×105) were seeded at the scaffold and scaffold@CNPs surface for 3 days. The cells were harvested by trypsin and washed twice by PBS. Samples were then suspended in cold 75% ethanol and kept at 4 °C overnight. After washing twice by PBS, the samples were added 100 µL PI (50 µg/mL) and incubated for 10 min. Finally, the cell cycles were detected on a BD FACS Calibur system and the results were analyzed by FlowJo software. 2.6. MSCs apoptosis analysis MSCs (1×104) were seeded at the scaffold and scaffold@CNPs surface for 3 days. The cells were harvested by trypsin and washed twice by cold PBS. Each sample was added 100 µL 1× 6


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binding buffer, 2 µL FITC-Annexin V and 2 µL PI. The samples were protected from light and incubated at room temperature for 15 min. Cell apoptosis was analyzed on a BD FACS Calibur system and the results were analyzed by FlowJo software. 2.7. qRT-PCR detection Total RNA of MSCs and EPCs were extracted by Trizol (Takara) and reverse transcribed to cDNA by PrimeScriptTM (Takara) with gDNA Eraser following the manufacturer's instructions. The primers were shown in Table S1. The real-time PCR was performed on a BIO-RAD CFX96 Real-Time System instrument using SYBR Premix Ex TaqTMⅡ(TaKaRa) as fluorescent probe. The cycling conditions were set as 95 °C for 30 seconds, 40 cycles of 95 °C for 5 seconds, and 60.5 °C for 34 seconds. All experiments were performed in triplicate. The relative mRNA level was calculated by the normalization to that of GAPDH. 2.8. EPCs positive ratio EPCs positive ratio was determined by FACS using CD34 and Flk-1 as surface markers. EPCs were co-cultured with MSCs for 7 and 14 days before running the test. The cells were detached by trypsin and washed by PBS for twice. FITC conjugated anti-mouse CD34 monoclonal antibody (eBioscience) and PE conjugated anti-mouse Flk-1 monoclonal antibody (eBioscience) was added to the cells according to the protocol provided by the manufacturer. After incubation at 4°C for 30 min, samples were washed by PBS plus 0.5% BSA. Rat IgG2a K Isotype control of FITC/PE was used to exclude the non-specific adsorption. After washing twice by PBS, the flow cytometric acquisitions were performed using a BD FACS Calibur system with a minimum of 10,000 events acquired for each sample. Live cells used for the analysis were gated based on forward angle light scatter (FSC) 7



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and side angle light scatter (SSC) characteristics. Finally, the EPCs positive ratio was analyzed by FlowJo software. 2.9. Immunofluorescence analysis EPCs separated from two mice spleen were cultured in 12 well plate for two days. The cells were washed with PBS to remove the non-adhesion cells. MSCs (1×104) were seeded on scaffold or scaffold@CNPs and cultured in the apical compartment of the transwell plate. Immunofluorescence detection was performed at 14 days. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 10 min prior to staining. The cells were then incubated with bovine serum albumin (BSA) 1% (w/v) for 30 min to avoid non-specific binding. After that the cells were incubated with primary anti-eNOS and anti-vWF antibody (1: 100 dilution, Santa Cruz) for 60 min. The primary antibody conjugated cells were washed for several times and then incubated with secondary antibody for 60 min (Cy3 red fluorescence for anti-vWF antibody and FITC green fluorescence for anti-eNOS antibody). The cells were washed extensively to remove the unconjugated dye. The nucleus was stained with Hoechst 33342 (1: 1000 dilution) for 20 min followed by extensive wash. Finally, the coverslips were mounted on microscope slides with the storage solution and sealed with mount gel. The images were gathered on a Leica TCS SP5 laser scanning confocal microscope. 2.10. ELISA analysis MSCs (1×104) were seeded on scaffold and scaffold@CNPs in 24 plate well. The quantification of CCL-2, CCL-3, IL-6 and VEGF secreted from MSCs were determined using ELISA kit

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(CUSABIO biotechnology, China) according to the manufacturer’s instruction. The amount of CCL-2, CCL-3, IL-6 and VEGF released in the supernatant was expressed in nanogram per liter. 2.11. VEGFR inhibition EPCs separated from two mice spleen were cultured in 12 well plate for two days. The cells were washed with PBS to remove the non-adhesion cells. The EPCs were co-cultured with MSCs in a non-contact manner. MSCs (1×104) were seeded on scaffold or scaffold@CNPs and cultured in the apical compartment of the transwell plate. EPCs cultured in the basolateral compartment were treated with 10µM JK-P3 in DMEM/F12 medium supplemented with 20% FBS. The EPCs were harvested at 14 days for western blot and CCK8 cell viability analysis. 2.12. Western blot analysis Proteins were extracted and electrophoresed on SDS-PAGE Gel. After separation, proteins were transferred onto nitrocellulose membrane sheets (GE Healthcare Japan). After that the membrane was blocked in 5% low-fat dry milk in TBS-T at room temperature for 1 h and incubated with primary antibodies including eNOS (rabbit polyclonal 1: 500, Abcam), vWF (goat polyclonal 1: 500, Santa Cruz), PECAM-1 (goat polyclonal 1: 500, Santa Cruz), HIF-1α (rabbit polyclonal 1: 500, Abcam), β-actin (mouse monoclonal 1: 1000, Cell Signaling Technology) at 37 °C overnight. Immunoreactive bands were visualized on a BIO-RAD ChemiDoc XRS+ system. 2.13. Endothelial tube formation assay EPCs were co-cultured with MSCs for 28 days before running the tube formation assay. Cells were seeded on Matrigel (BD Bioscience) coated 24 well plate at density of 1×105 cells per well in DMEM/F12 media complemented with 20% FBS for 5 h at 37 °C. After washing by PBS, the cells 9



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were fixed with 4% paraformaldehyde. The images were taken on an IX71 Olympus microscope and the branch points quantifications were analyzed on Image J. 2.14. Intracellular free Ca2+ detection MSCs were cultured on scaffold and scaffold@CNPs for 1, 7 and 14 days. The cells were harvested by trypsin and washed by Hanks' Balanced Salt Solution. After that the cells were suspended in Fluo-3 AM (5 µM) for 45 min in dark. Fluo-3 AM can easily pass the cell membrane and cut into Fluo-3 by intracellular esterase. The Fluo-3 then specifically combines with Ca2+ that emits strong fluorescence under the excitation wavelength of 488 nm. Based on this principle, the detection of intracellular Ca2+ was carried out by flow cytometer at 488 nm excitation wavelength. 2.15. Animal experiment The experimental protocol was approved by the Animal Care and Use Committee of Third Military Medical University (China). Each of the scaffold and scaffold@CNPs were seeded with MSCs (5×105) and cultured in vitro for 2h. Nude mice with average body weight of 18g were anaesthetized by pentobarbital sodium (75mg/kg body weight). Under sterile condition, four incisions were made at the back of the mouse near the limbs. The MSCs loaded scaffold and scaffold@CNPs were implanted at the left and right side incisions of the mouse respectively. The grafts were taken out six and twelve weeks after implantation and the samples were fixed in 4% paraformaldehyde for 24h. Samples were decalcified in PBS containing 10% EDTA with pH of 9.0 for one month. After that, samples were dehydrated and embedded by paraffin, and subsequently cut to sections with thickness of 5 µm for further analysis. HE and masson's trichrome staining were performed separately on tissue sections according to the manufacturer's protocols, and images were 10


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captured under microscope. For CD31 immunohistochemistry staining, tissue slides were deparaffinized, rehydrated and submerged in hydrogen peroxide to quench peroxidase activity. The slides were then incubated with 1% BSA before exposure to primary antibody against CD31 (1:50, Abcam, ab28364). After incubation at 4°C overnight, HRP conjugated secondary antibody was applied to the slides for 1 h at room temperature. Diaminobenzidine (DAB, Beyotime, China) kit was used to develop the color and the slides were counterstained with hematoxylin. Finally, the slides were imaged using microscope. 2.16. Statistical analysis Statistical significance was determined using IBM SPSS statistics (21) and a statistical difference was considered significant when the *p

Cerium Oxide Nanoparticle Modified Scaffold ... - ACS Publications

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