Copyright © 2014 Cognizant Communication Corporation DOI: 10.3727/096368914X681018 CT-1191 Accepted 02/19/2014 for publication in “Cell Transplantation”
In situ recruitment of human BMSCs using chemokines for articular cartilage regeneration Min Sung Park1,2, Yun Hee Kim2, Youngmee Jung3, Soo Hyun Kim3, Jong Chul Park4, Dong Suk Yoon1,2, Sung-hwan Kim2, Jin Woo Lee1,2* 1
Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, South Korea
2
Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, South Korea
3
Biomaterials Research Center, Korea Institute of Science and Technology, Seoul, South Korea
4
Department of Medical Engineering, Yonsei University College of Medicine, Seoul, South Korea
*Corresponding Author: Jin Woo Lee, M.D., Ph.D. Department of Orthopaedic Surgery-29 Yonsei University College of Medicine, Seoul 120-752, South Korea Phone: 82-2-2228-2190 Fax: 82-2-363-1139 E-mail:
[email protected]
Running title: BMSCs homing promising OA repair
CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation
ABSTRACT Bone marrow-derived mesenchymal stem cells (BMSCs) are a good cell source for regeneration of cartilage as they can migrate directly to the site of cartilage injury and differentiate into articular chondrocytes. Articular cartilage defects do not heal completely due to the lack of chondrocytes or BMSCs at the site of injury. In this study, the chemotaxis of BMSCs toward chemokines, which may give rise to a complete regeneration of the articular cartilage, was investigated. CCR2, CCR4, CCR6, CXCR1, and CXCR2 were expressed in normal BMSCs and were increased significantly upon treatment with proinflammatory cytokines. BMSC migration was increased by MIP-3α and IL-8 more than by MCP-1 or SDF-1α. IL-8 and MIP-3α significantly enhanced the chemotaxis of BMSCs compared with MCP-1, SDF-1α, or the PBS. Human BMSC recruitment to transplanted scaffolds containing either IL-8 or MIP-3α significantly increased in vivo compared with that to scaffolds containing the PBS. Furthermore, IL-8- and MIP-3α-containing scaffolds enhanced tissue regeneration of ostechondral defect site formed in beagle knee articular cartilage. Therefore, this study suggests that IL-8 and MIP-3α are the candidates that induce the regeneration of damaged articular cartilage.
Key Words: Chemokines, IL-8, MIP-3α, Mesenchymal stem cells
CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation
INTRODUCTION An articular cartilage injury causes severe chronic pain and decreased mobility. Several surgical techniques have been widely used to repair injured articular cartilage. Subchondral drilling is a simple method, but the articular cartilage defects are filled with fibrocartilage. Osteochondral autograft transplantation (OAT) is a promising method to treat articular cartilage injuries, but donors are limited. Although autologous chondrocytes transplantation (ACT) is another good treatment modality, ex vivo cell expansion is required and the treatment can be both expensive and difficult (6,7,39). To overcome these problems, tissue engineering using biodegradable materials has been attempted to successfully repair damaged articular cartilage (41). Autologous chondrocytes or mesenchymal stem cells (MSCs) seeding on implanted scaffolds is necessary for the successful regeneration of damaged articular cartilage because tissue engineering using scaffolds alone is insufficient to avoid the current problems (1). Cartilage regeneration using autologous cells can be accomplished by the natural progression of cartilage development (17). Autologous MSCs have been highly regarded as an alternative cell source for the repair of articular cartilage defects as autologous MSCs can differentiate into chondrocytes and osteocytes under tissue-specific culture conditions (42). The ex vivo expansion of MSCs is required for cartilage regeneration. However, the loss of osteogenic or chondrogenic potential of primary MSCs and the secondary surgery for cultured cell implantation remain as problems in this therapeutic strategy (34). Therefore, a new strategy should be developed to overcome the current problems and successfully repair articular cartilage using autologous MSCs. A chemokine is a chemotactic cytokine that plays a crucial role in cell recruitment. Recent studies have demonstrated that chemotaxis of bone marrow-derived MSCs (BMSCs) CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation is induced by chemokines and repairs the wounded tissue (29,37). If this concept is applied to cartilage repair, the in situ recruitment of BMSCs to the site of defect using chemokines may be an effective method for inducing cartilage tissue regeneration. Unfortunately, few reports of articular cartilage tissue regeneration using chemokine-recruited BMSCs exist. Therefore, our purpose was to identify the chemokines that trigger autologous BMSCs to be sent from a bone marrow niche to the site of articular cartilage damage for cartilage regeneration.
MATERIALS AND METHODS Cell culture Human bone marrow was aspirated from the posterior iliac crest of 8 healthy male and female donors aged 13-60 years with approval of the Institutional Review Board (IRB) of our institution, and written informed consent was obtained from all patients. BMSCs were isolated based on their capacity to adhere to tissue culture plastic, and the isolated cells were kept separately by each donor. BMSCs were cultured in DMEM-low glucose (DMEM-LG, GIBCO-BRL, Grand Island, NY, USA) containing 10% FBS, GIBCO BRL, and an 1X antibiotic-antimycotic solution (GIBCO BRL). BMSCs (passage 2-4) were used in all experiments.
In vitro mimicking of articular cartilage injury After serum starvation of BMSCs (serum-free DMEM-LG containing 1X antibioticantimycotic solution,1%(v/v) insulin, transferrin and selenium supplement (ITS, GIBCOBRL)) for 24 hours, cells were stimulated by adding 10 ng/mL of interleukin-1beta (IL-1β, R&D Systems Inc., Minneapolis, MN, USA) or 10 ng/mL of tumor necrosis factor-alpha (TNF-α, R&D Systems Inc.) to the media for 4, 24, or 48 hours. The stimulated cells were CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation washed with phosphate-based saline (PBS) solution (GIBCO-BRL) twice and then used for our experiments.
Reverse dot-blot hybridization Sample DNAs of 19 chemokine receptors, TATA box-binding protein (TBP), 18S rRNA (as a positive control), and rabbit GAPDH (as a non-specific negative control) were amplified using RT-PCR (Table 1). Probe cDNAs were synthesized from IL-1β or TNF-α-stimulated human BMSCs. Sample DNAs were spotted onto Hybond-N+ nylon membranes (Amersham Phamrmacia Biotech). The probe cDNAs were labeled using γ32P-dCTP (NEN Life Sciences, Boston, USA). The membranes were hybridized with the probe cDNAs for 15 hours and then washed in 2X saline-sodium citrate (SSC; Sigma, St. Louis, MO, USA) /0.1% sodium dodecyl sulfate (SDS; Sigma). Hybridized probes were acquired with an integrated imaging plate (IP, Fujifilm, Tokyo, Japan) and detected using a fluorescent image analyzer (FLA7000, Fujifilm). Images of hybridized samples were quantified by densitometry (TINA pixel analyzer version 2.10, Raytest Isotopenme gerate GmbH, Straubenhardt, Germany).
Wound healing assay BMSCs were plated at 2,000 cells per well on silicon culture inserts (Ibidi, Integrated BioDiagnostics, Munich, Germany) attached to 24-well tissue culture plates (SPL, Pocheon, South Korea) in DMEM-LG containing 10% FBS. The cells were incubated until they reached ~80% confluence, and the culture inserts were then removed. The cells were gently washed in PBS. BMSCs were treated with 500 ng/mL of interleukin-8 (IL-8, R&D Systems Inc.), stromal cell-derived factor-1alpha (SDF-1α, R&D Systems Inc.), monocyte chemotactic protein-1 (MCP-1, R&D Systems Inc.), and macrophage inflammatory protein-3alpha (MIP3α, R&D Systems Inc.) in serum-free media. Cells that migrated to the wounded area CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation (diameter=4 mm) were photographed and counted before and after chemokine treatment for 1, 3, 5, 7, 9, and 12 hours. Cells were counted in triplicate by three independent observers.
Real-time in vitro cell tracking assay BMSCs were grown to ~80% confluence in silicon culture inserts on glass side disks (Live Cell Instrument, Inc. (LCI), Nowon-gu, Seoul, Korea) in DMEM-LG containing 10% FBS. After serum-starvation of BMSCs for 24 hours, the culture inserts were removed from the glass disks. The cells were gently washed in PBS, and 500 ng/mL of chemokine (IL-8, SDF-1α, MCP-1, and MIP-3α) was added to BMSCs in each well containing serum-free media. To track BMSCs moving into the gap (diameter=4mm), cell-attached glass slides were incubated in 35-mm dish magnetic chambers (Chamlide™, LCI) and the migration of BMSCs was recorded using a microscope (CK-41, Olympus, Tokyo, Japan) equipped with a CCD camera (KP-D20BU, HITACHI, Tokyo, Japan) and capture software (TOMORO version FG 2.9.19, Olympus) every 5 min for 48 hours. The total distance of cell migration and the average cell migration velocity were calculated using Image J software (version 1.44, http://imagej.nih.gov/ij/, NIH, Maryland, USA). The experiment was performed in triplicate.
In vitro chemotaxis assay BMSCs were serum-starved for 24 hours and inoculated at 10,000 cells per 200 μL of serum-free media into each 24-well culture insert system (8-μm pores) (Falcon; Becton, Dickinson and Company, Heidelberg, Germany). A volume of 400 μL of serum-free media containing 0.1% BSA (Sigma) or 500 ng/mL of IL-8 or MIP-3α was added into the outside of each insert, and the cells were incubated for 6 hours. After incubation, the cells were washed in PBS and stained with crystal violet solution (Merck KGaA, Darmstadt, Germany). The non-migratory cells were removed with a cotton-tipped swab (Millipore, Billerica, MA, USA) CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation and stained cells (attached on the outside of the inserts) were counted using a microscope. Cell counting was performed in triplicate by three observers.
Osteogenic or chondrogenic differentiation of human BMSCs After BMSCs were grown to confluence in growth medium, osteogenic differentiation was achieved with cultures in a basic medium containing 100 nM of dexamethasone (Sigma), 50 μM of ascorbic acid-2-phosphate (Sigma), and 10 mM of β-glycerophosphate (Sigma), and 10ng/ml of IL-8 and MIP-3α each for 14 days. Ascorbic acid and the two chemokines were added daily and the osteogenic differentiation medium was changed every 2-3 days. The osteoblast differentiation was confirmed by alkaline phosphatase (ALP) and von kossa stainings. For ALP staining, the cells were stained with an alkaline dye solution (Sigma) for 30 min in the dark and counterstained with Mayer’s hematoxylin solution (Sigma). For von kossa staining, the cells were incubated in fresh 3% silver nitrate (Sigma). The experiment was performed in triplicate. For chondrogenesis, BMSCs were dotted for 4 hours at 8,000 cells per 10 μL of growth medium in each culture well. Chondrogenic differentiation was induced with cultures in DMEM-high glucose containing 50 μM of ascorbic acid-2-phosphate, 10 ng/ml of transforming growth factor-beta 3 (TGF-β3, R&D Systems Inc.), and 10 ng/ml of IL-8 and MIP-3α each for 21 days. Ascorbic acid, TGF-β3, and the two chemokines were added daily. Chondrogenesis was confirmed by hematoxylin and eosin (H&E) staining, masson’s trichrome staining, and RT-PCR for type II collagen A1 and aggrecan (Table 1). Differentiated micromasses were embedded in paraffin and stained with H&E and with Masson’s trichrome solution (Sigma) to examine collagen fiber formation. The integrated optical density of total collagen in the micromass was measured using a microscope equipped with a CCD camera (BX-51, Olympus) and analyzed using MetaMorph® Microscopy CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation Automation & Image Analysis Software (version 7.7.1.0, Molecular Devices Inc. Sunnyvale, CA, USA). The experiment was performed in triplicate.
In vivo chemotaxis assay An in vivo chemotaxis assay was performed in male nude mice by injecting human BMSCs and transplanted poly-lactide-co-glycolide (PLGA) scaffolds containing either chemokines or PBS. This experiment was conducted in accordance with the ethical guidelines for the Care and Use of Laboratory Animals of the Yonsei Biomedical Research Institute Committee. 1) Complexation of the scaffolds and the chemokines PLGA scaffolds (porosity=70%, pore size=300~500 μm, diameter=5 mm, thickness=4 mm, disk type) were used as a chemokine carrier (21). Briefly, The PLGA (poly D,L-lactideco-glycolic acid, MW = 97,000, Resomer; Sigma) scaffold was prepared by the novel sintering method. PLGA, and NaCl (300-500㎛, 70% w/w) were homogeneously mixed and
put into disk mold, the mixture was pressed at 150MPa for 2min and baked at 180℃ for 35min. For the salt removing, it was leached in deionized water with shaking for 7days. Next, the scaffold (diameter=5mm / thickness=4mm) were freeze-dried for 48h and sterilized by ethylene oxide gas. The scaffolds were soaked by repeated pipetting in PBS containing 500 ng/mL of IL-8 or 500 ng/mL of MIP-3α for 24 hours at 4°C. PBS- or chemokine-containing scaffolds were then subcutaneously transplanted into the back of nude mice. 2) Cell labeling with NIR-nanoparticles Near-infrared
(NIR)
nanoparticle
labeling
(NEO-LIVE
Magnoxide
675,
Extension/Emission (Ex/Em)=675/700 nm, Biterials Co., Ltd., Itaewon-dong, Seoul, Korea) was performed according to the manufacturer’s instructions. Briefly, human BMSCs were CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation incubated until they reached a confluence of ~80%. NIR-nano particles were collected by centrifugation at 13,000 X g for 10 min and washed with PBS twice and added to cells in DMEM-LG containing 10% FBS at a ratio of 4 mg/mL of growth medium. Then cells were incubated at 37°C, 5% CO 2 for 48 hours. After labeling, cells were washed with PBS twice and collected by trypsinization. After labeling, cells were washed with PBS and collected by trypsinization. Cell labeling was confirmed using a multi-spectrum in vivo fluorescence imaging system (Maestro 2, Cambridge Research & Instrumentation (CRi) Inc., MA, USA). 3) Real-time in vivo fluorescence imaging assay Carrier free-chemokine containing scaffolds and NIR-labeled human BMSCs were implanted into twenty balb/c male nude mice (Orient Bio Inc., Seongnam, South Korea). A total of 2X106 BMSCs were injected into the tail veins, and scaffolds containing IL-8 or MIP3α were subcutaneously transplanted into the back of the mice. After scaffold transplantation, the skin was closed using black nylon sutures (Ethicon Inc., Somerville, New Jersey, USA). The movement of injected cells was tracked in real-time every 2-3 days using a multispectrum in vivo fluorescence imaging system (Cambridge Research & Instrumentation (CRi) Inc.). The images were quantified using Maestro 2 software (Maestro2 Work Area, Cambridge Research & Instrumentation (CRi) Inc., MA, USA). All mice were euthanized and all scaffolds were harvested at six weeks. At necropsy, inserted scaffolds were closely examined, photographed and harvested at six weeks. The scaffolds and adjacent tissues were evaluated for subdermal tissue formation and vascular infiltration. This experiment was performed at least in triplicate.
Confocal laser scanning microscopy Detailed observation using a laser scanning microscope (LSM 700, Carl Zeiss MicroImaging GmbH, Jena, Germany) was performed to confirm the presence of the injected CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation NIR-labeled human BMSCs in the chemokine/scaffold complexes. All 5-μm thick sections on the glass slides were stained with eosin solution and mounted to identify the cell nucleus. Eosin-stained sections were observed using laser scanning microscope with a laser beam of Ex=630 nm/Em=700nm (Carl Zeiss).
Immunohistofluorescence analysis Immunohistochemical reactions using mouse anti-CD11b-FITC and CD45 (Abcam, Cambridge, UK) were performed. All sections were treated with 3% H 2 O 2 (Duksan Pure Chemicals, Ansan, South Korea) for 10 min to inactivate endogenous peroxidase. Then, the sections were incubated with anti-CD11b-FITC (dilution factor=1:100) or CD45 (dilution factor=1:50) primary antibodies overnight. The sections were incubated with FITCconjugated secondary antibody for 30 min at room temperature. After counterstaining with hematoxylin, the sections were mounted, and the images were acquired using an inverted fluorescence microscope (IX-71, Olympus) and a digital camera (DP-71, Olympus).
Preparation of biodegradable biphasic scaffold for osteochondral defect model of beagle The PLA (Poly lactic acid)/β-TCP (Poly-L-lactide/beta-tricalciumphosphate) scaffolds for the bone part were prepared with PLA powder(MW = 245,000; CM research, Korea) and β-TCP (100~300㎛; Berkeley Advanced Biomaterials, CA, USA) mixed with NaCl (diameter = 300~500㎛; Sigma) by the novel sintering method. And PLCL(poly L-lactide-co-εcaprolactone, MW = 328,000) was synthesized and used in a gel-pressing method to fabricate scaffolds for the cartilage part. After PLA, β-TCP, and NaCl particles (300~500㎛, 70% w/w) were homogeneously mixed and placed in a cylindrical mold (diameter = 5.85mm), the CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation mixture was pressed at 150MPa for 2min at room temperature using a hydraulic press to yield solid block and further treated at 210℃ for 30min. PLCL synthesized in our laboratory was
dissolved in tetrahydrofuran (THF) (5%w/v) and then mixed with NaCl particles (300~500㎛, 85% w/w). The THF was then evaporated in air to form the PLCL gels, which were subsequently pressed on the PLA/β-TCP blocks prepared above to combine two parts in a cylindrical mold(diameter = 6mm). Next, the residual THF was evaporated for 24h at room temperature, after which it was completely removed under vacuum for 72h. The salts were then leached out by placing the gels in distilled, deionized water with constant shaking for 7 days. The resulting scaffolds were then freeze-dried for 48h, after which they were sterilized with ethylene oxide gas. The final product has 8mm in length (PLA/β-TCP = 7mm, PLCL = 1mm) and 6mm in diameter.
Surgical procedures and histological analysis in osteochondral defect model of beagle Male beagles (Orient Bio Inc.) were used as experimental animal models (n=12) having a weight of constant 14 ~ 15 kg the same week of age to overcome individual variations. This experiment was also conducted in accordance with the ethical guidelines for the Care and Use of Laboratory Animals of the Yonsei Biomedical Research Institute Committee. Under general anesthesia using 2% isoflurane (Sigma), a medial parapatellar incision was made over the right knee and the patellar was everted. The 6-mm-diameter and 8-mm deep cylindrical osteochondral defect, identical to the dimension of a biphasic scaffold, was made using an Osteochondral Autograft Transfer System (OATS, Arthrex, Naples FL, USA) in the weightbearing articular surface of the femoral medial condyle. The biodegradable biphasic scaffold composed of poly-L-lactide acid/β-tricalcium phosphate (PLLA/β-TCP) was press-fitted into the defect of the knee and the surface of repaired defect flushed smoothly with the native CT-1191 Cell Transplantation Epub; provisional acceptance 02/07/2014
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Copyright © 2014 Cognizant Communication Corporation articular surface. Each scaffold containing 5ug of IL-8 or MIP-3α was inserted into osteochondral defect site to evaluate the regeneration potential of the damaged bone and cartilage.
Statistical analysis All data are represented as means ± standard deviation (SD)≥3). (n The experimental groups were compared with the control group in triplicate. Statistical significance was determined using paired student’s t-tests, ANOVA, and post-hoc tests (pair-wise comparisons) with a Bonferroni adjustment to control for errors according to experimental methods. Significance was defined as p values
