REGENERATIVE MEDICINE Mesenchymal Stem Cells Reduce Intervertebral Disc Fibrosis and Facilitate Repair

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Department of Orthopaedics & Traumatology; b Department of Biochemistry; cCentre for Reproduction, Development, and Growth; eDepartment of Mechanical Engineering, and i Department of Electrical & Electronic Engineering, The University of Hong Kong, Hong Kong SAR, People’s Republic of China; f Department of Micro-nano Materials and Devices, South University of Science and Technology of China, Guangzhou, People’s Republic of China; d Mechanobiology Institute, Singapore; gDepartment of Bioengineering, and h Department of Mechanical Engineering, National University of Singapore, Singapore; jDepartment of Orthopaedic Surgery, University of California, San Diego, California, USA Correspondence: Kenneth M.C. Cheung, M.D., Department of Orthopaedics and Traumatology, The University of Hong Kong Medical Centre, Queen Mary Hospital, Pokfulam Road, Pokfulam, Hong Kong SAR, People’s Republic of China. Telephone: 1852-2855-4254; Fax: 1852-2817-4392; e-mail: [email protected]; or Danny Chan, Ph.D., Department of Biochemistry, 3/F, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong SAR, People’s Republic of China. Telephone: 18522819-9482; Fax: 1852-28551254; e-mail: [email protected] Received September 30, 2013; accepted for publication March 20, 2014; first published online in STEM CELLS EXPRESS April 15, 2014. C AlphaMed Press V

1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.1717

VICTOR Y.L. LEUNG,a,b,c DARWESH M.K. ALADIN,a,d FENGJUAN LV,a VIVIAN TAM,a YI SUN,a ROY Y.C. LAU,a SIU-CHUN HUNG,a ALFONSO H.W. NGAN,e BIN TANG,f CHWEE TECK LIM,d,g,h ED X. WU,i KEITH D.K. LUK,a WILLIAM W. LU,a KOICHI MASUDA,j DANNY CHAN,b,c KENNETH M.C. CHEUNGa,c Key Words. Disc degeneration • Mesenchymal stem cells/multipotent stromal cells • Fibrosis Collagen fibril • Matrix metalloproteinase 12 • Fibrillogenesis

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ABSTRACT Intervertebral disc degeneration is associated with back pain and radiculopathy which, being a leading cause of disability, seriously affects the quality of life and presents a hefty burden to society. There is no effective intervention for the disease and the etiology remains unclear. Here, we show that disc degeneration exhibits features of fibrosis in humans and confirmed this in a puncture-induced disc degeneration (PDD) model in rabbit. Implantation of bone marrow-derived mesenchymal stem cells (MSCs) to PDD discs can inhibit fibrosis in the nucleus pulposus with effective preservation of mechanical properties and overall spinal function. We showed that the presence of MSCs can suppress abnormal deposition of collagen I in the nucleus pulposus, modulating profibrotic mediators MMP12 and HSP47, thus reducing collagen aggregation and maintaining proper fibrillar properties and function. As collagen fibrils can regulate progenitor cell activities, our finding provides new insight to the limited self-repair capability of the intervertebral disc and importantly the mechanism by which MSCs may potentiate tissue regeneration through regulating collagen fibrillogenesis in the context of fibrotic diseases. STEM CELLS 2014;32:2164–2177

INTRODUCTION Intervertebral discs (IVD) are semigelatinous/cartilaginous connective tissues that play a crucial role in spinal column articulation, force coordination, and cushioning against axial load. The degeneration of IVD, also known as degenerative disc disease, is suggested to share certain pathophysiological characteristics of osteoarthritis [1]. Disc degeneration is one of the most common disorders reported in orthopedic practice and contributes to neck and back pain, which is the leading cause of disability in people aged less than 45 years with an overall cost exceeding $100 billion per year in the United States for lost wages and productivity [2, 3]. Disc degeneration prevalence progresses linearly with increase in age. By the age of 60 years, 100% of subjects exhibit some form of disc degeneration [2]. The etiology and, in particular, what causes the irreversible progression of degeneration and lack of self-repair is still poorly understood. Conventional treatments aim to alleviate symptoms by surgeries such as disc excision and fusion of spinal segments, which however, may result in impaired spinal kinematics and juxta level degeneration.

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Fibrosis is common to chronic inflammatory conditions and is related to excessive tissue remodeling as a result of disrupted wound healing [4]. Abnormal deposition and/or crosslinking of the collagen matrix ultimately leads to defective cellular homeostasis and repair, thence to hardening and scarring of tissues [5, 6]. Anatomical studies on autopsy and surgical specimens show evidence of fibrosis in the majority of degenerated discs [7–9]. Our previous study has also shown abnormal collagen fibril bundling in mechanically compromised discs [10], suggesting that abnormal collagen matrix meshwork is one of the major factors for reduced elastic and viscous behaviors of the disc that ultimately leads to altered kinematics of the joint under load. To date, there are no effective antifibrotic drugs that can ameliorate fibrotic diseases. Implantation studies in remnant kidney, bleomycin-induced lung injury, and myocardial infarction models suggest that mesenchymal stem cells (also known as mesenchymal stromal cells and multipotent stromal cells, or MSCs) possess antifibrotic activities [11–13]. MSCs are also shown to reduce disc degeneration in various animal models [14], and MSC-based disc C AlphaMed Press 2014 V

Leung, Aladin, Lv et al. regeneration therapy alleviated low back pain and neurologic symptoms in a pioneer clinical study [15]. We therefore sought to define how fibrosis is linked to disc homeostasis and degeneration and explore how MSCs could be used to ameliorate this. We here report that MSCs are capable of arresting disc degeneration through regulating fibrogenic events, leading to significant functional and structural recovery witnessed from the nano-to-macro scale. Our combined mechanical and biological data implicate an unidentified role of fibrillogenesis in disc degeneration and MSC-assisted repair. Our study sheds light on a function of MSCs in potentiating regeneration of tissue involved in fibrotic diseases.

MATERIALS

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METHODS

All animal experiment protocols were approved by the local government agency (Department of Health, Hong Kong SAR) and institutional ethics committee (CULATR). Use of human samples was approved by an Institutional Review Board with patient consent.

Patient Samples Degenerated lumbar disc tissues were collected from symptomatic patients undergoing disc excision and spinal fusion surgery. The samples were classified as grade 3 degeneration on the Schneiderman’s scale (which represents hypointense nucleus pulposus (NP) and disc space narrowing in magnetic resonance imaging (MRI)). Nondegenerated disc samples were collected from the lumbar discs of scoliosis patients undergoing deformity correction surgery.

Isolation and Culture of MSCs Human MSCs were isolated from the bone marrow of patients undergoing spinal fusion surgery. Cells were isolated within a few hours of bone marrow collection. Aspirates collected from the bone marrow were diluted 1:4 in Hanks’ balanced salt solution (Sigma-Aldrich, St. Louis, http://www. sigmaaldrich.com) and fractionated using Ficoll-Histopaque 1077 (1.077 g/ml, Sigma-Aldrich), according to the manufacturer’s manual. The mononuclear cell fraction was cultured in alpha-minimum essential medium (MEM) supplemented with 15% fetal calf serum, 1% penicillin/streptomycin, 1% L-glutamine, and 0.4% fungizone. The cells were maintained in a humidified incubator at 37 C with 5% CO2 in air with medium changed twice a week. The MSCs were confirmed to be CD45–, CD14–, CD34–, CD731, CD901, CD1051, and Stro-1low by flow cytometry analysis, and their potency in chondrogenic, osteogenic, and adipogenic differentiation was validated using STEMPRO differentiation kits (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The cells were used for subsequent experiments at passage 3–6. Bone marrow MSCs from 4 to 6 month old Chinchilla Bastard (ChB) rabbits (non-albino, outbred; Charles River Lab, Germany) were similarly isolated by the Ficoll method and cultured in complete DMEM (high glucose Dulbecco’s modified Eagle’s medium with 10% fetal calf serum, 20 mM HEPES, 22 mM sodium bicarbonate, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin) as previously described [16], and their multipotency was validated using STEMPRO differentiation kits (Invitrogen) at passage 3. MSCs

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at passage 2 or 3 were labeled by Qtracker 565 Cell Labeling Kit (Invitrogen) [17, 18] at a labeling efficiency >90%, then encapsulated in 0.25% PuraMatrix peptide hydrogel (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) as described by the manufacturer at 5 3 102 to 1.5 3 104 cells per ml for implantation experiments. Unlabeled MSCs were prepared similarly to evaluate effects of dosage and stage of application in long term implantation tests. To test the biocompatibility between MSCs and the hydrogel, the composite was cultured overnight then subjected to LIVE/DEAD cell viability assays (Invitrogen) as described by the manufacturer. Signals were captured under Eclipse 80i imaging system (Nikon, Japan, http://www.nikon.com).

Animal Model Lumbar disc degeneration (L2/L3 and L4/L5) was induced by annulus puncture with a 19G needle (BD Medical, Franklin Lakes, NJ, http://www.bd.com) through an anterolateral retroperitoneal approach in 6-month old New Zealand White (NZW) rabbits (inbred; Charles River Lab, Canada, http://www.criver.com) under anesthesia as previously described [19, 20]. The discs were assigned to either mild or severe degeneration group: mild degeneration was generated by a swift puncture at a 5-mm depth; severe degeneration was generated by puncturing at a 5mm depth with one cycle of needle rotation along the needle axis inside the disc. The disc degeneration status was evaluated by MRI, where the severity of the degeneration was determined by %DHDi (mild, 210 to 240 %DHDi; severe, 241 to 270 %DHDi) at 4 weeks postpuncture. Disc levels with a %DHDi value not within the defined range were not used in subsequent experiments. The operated animals were randomly divided into groups of four rabbits (i.e., total eight experimental discs in each group). For the MSC group, Qtracker-labeled MSC-hydrogel composite was injected into the NP of the degenerated NZW rabbit discs using the same surgical approach at 5 3 103 cells per disc in a volume of 10 ml using a 27G hypodermic needle (Hamilton, Reno, NV, http://www.hamiltoncompany.com). For control groups, 10 ml of phosphate buffered saline (PBS) or hydrogel was injected. At 4 and 12 weeks post-treatment, disc hydration was quantified by MRI and disc height was determined from radiographs. For long-term evaluation, unlabeled MSC-hydrogel composites were implanted at 5 3 103 to 1.5 3 105 cells in a 10 ml volume, and then the disc hydration and height were followed at 1- and 3-month intervals up to 1 year post-treatment. No immunosuppressant was applied after implantation in any of the animals.

Imaging The animals were anesthetized and sagittal T2-weighted images of their lumbar spines were acquired by 3T Siemens Magnetom Trio scanner (Siemens, Munich, Germany, http://www.sie mens.com) together with 0%–100% standards of deuterium oxide:hydrogen oxide (H2O:D2O). The T2 signal of the disc to be tested was quantified as H2O:D2O index (HDi) as previously described [21] and the values were expressed as %HDi using intrasubject L3/4 level as reference, where %HDi 5 (HDitest/ HDiref) 3 100%. Percentage change in %HDi (%DHDi) was calculated, where %DHDi 5 %HDi – 100%. Lateral radiographs of the lumbar spine were taken using cabinet X-ray system (Faxitron Corp, Tucson, AZ, http://www.faxitron.com). The disc C AlphaMed Press 2014 V

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height index of the disc levels from L2 to L5 was calculated as previously described [20] and expressed as % disc height index using intrasubject L3/4 level as reference.

Identification of Implanted MSCs The endplate-disc-endplate segments were harvested, frozen, and embedded in OCT compound (Sakura Tissue, Netherlands, http://www.sakura.eu). Cross-sections were obtained at 20 mm thickness using a cryotome (Model CM1510S, Leica, http://www.leicabiosystems.com). The sections were stained with 4’,6-diamidino-2-phenylindole (DAPI) and visualized by fluorescence microscopy using a Nikon Eclipse 80i imaging system at 488 nm to identify the Qtracker-labeled MSCs and 360 nm for DAPI stained nuclei. Average quantities of NP cells (blue) and MSCs (green) from three mid-sections were determined using Image-Pro Plus 2.0 (Media Cybernetics, Crofton, MD, http://www.mediacy.com), and the mean was calculated from four independent disc samples.

Motion Stiffness and Confined Compression Analysis Spinal segments were harvested at 12 weeks post-treatment for testing by mechanical loading. Macroscale lateral bending, flexion and extension stiffness, as well as axial rotation stiffness of the L2/3 and L4/5 spinal segments without the influence of posterior elements (facet joints and posterior longitudinal ligament), were tested on an 858 Mini Bionix testing system (MTS system, Eden Prairie, MN, http://www. mts.com) as previously described [22]. Briefly, the bending stiffness, expressed as force/displacement, was determined at the early and late phase of displacement at the fifth loading cycle. Extension stiffness was measured only at the late phase. The rotational stiffness was determined at the fifth loading cycle and expressed as torque (force 3 displacement/ degree of rotation). After testing the motion segment as a whole, the vertebral body and endplate from one end of the disc was carefully removed to expose the NP. An NP specimen of 3 mm in diameter was excised from the center of each sample. Microscale confined compression testing was performed using a MicroTester (Model 5948, Instron, Norwood, MA, http://www. instron/com) by modifying a previously established experimental setup and protocol for testing human NP tissues [10, 23]. Briefly, the specimen was placed into a confining chamber and loaded by a porous platen at a stable tare load of 0.02–0.03 N, followed by measurement of the tissue thickness. An isometric swelling test was performed, where the chamber was filled with PBS at room temperature. After 1 minute, a 0.1% compressive strain (ramp rate, 0.25 lm/second) was applied, followed by a 30-minute hold period (swelling phase), and then a 10% compressive strain (ramp rate, 0.25 lm/second). Two parameters, the swelling pressure, Psw, measured as the equilibrium stress reached by the end of the swelling phase, and the compressive modulus, K, calculated from the slope of the stress versus strain plot during the application of 10% compressive strain were calculated [24].

Scanning Electron Microscopy and Fibril Analysis NP sections of 8 lm thickness were adhered to Histobond1 microscope slides (Marienfeld, Heidelberg, Germany, http:// www.marienfeld-superior.com), gently rinsed in distilled water, and dried in a desiccator overnight at room temperature. The C AlphaMed Press 2014 V

Disc Repair by MSCs via Fibrosis Inhibition

dried samples were sputter-coated with a thin layer of gold (