Mesenchymal Stem Cells Reduce Intervertebral Disc Fibrosis and Facilitate Repair

Authors

  • Victor Y.L. Leung,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
    2. Department of Biochemistry, The University of Hong Kong, Hong Kong SAR, People's Republic of China
    3. Centre for Reproduction, Development, and Growth, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Darwesh M.K. Aladin,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
    2. Mechanobiology Institute, National University of Singapore, Singapore
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  • Fengjuan Lv,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Vivian Tam,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Yi Sun,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Roy Y.C. Lau,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Siu-Chun Hung,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Alfonso H.W. Ngan,

    1. Department of Mechanical Engineering, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Bin Tang,

    1. Department of Micro-nano Materials and Devices, South University of Science and Technology of China, Guangzhou, People's Republic of China
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  • Chwee Teck Lim,

    1. Mechanobiology Institute, National University of Singapore, Singapore
    2. Department of Bioengineering, National University of Singapore, Singapore
    3. Department of Mechanical Engineering, National University of Singapore, Singapore
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  • Ed X. Wu,

    1. Department of Electrical & Electronic Engineering, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Keith D.K. Luk,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • William W. Lu,

    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
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  • Koichi Masuda,

    1. Department of Orthopaedic Surgery, University of California, San Diego, California, USA
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  • Danny Chan,

    Corresponding author
    1. Department of Biochemistry, The University of Hong Kong, Hong Kong SAR, People's Republic of China
    2. Centre for Reproduction, Development, and Growth, The University of Hong Kong, Hong Kong SAR, People's Republic of China
    • 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: +852-2855-4254; Fax: +852-2817-4392; e-mail: cheungmc@hku.hk; 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: +852-2819-9482; Fax: +852-2855-1254; e-mail: chand@hku.hk

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  • Kenneth M.C. Cheung

    Corresponding author
    1. Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, People's Republic of China
    2. Centre for Reproduction, Development, and Growth, The University of Hong Kong, Hong Kong SAR, People's Republic of China
    • 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: +852-2855-4254; Fax: +852-2817-4392; e-mail: cheungmc@hku.hk; 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: +852-2819-9482; Fax: +852-2855-1254; e-mail: chand@hku.hk

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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 co-ordination, 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.

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 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 and 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, CD73+, CD90+, CD105+, 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 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 × 102 to 1.5 × 104 cells per µl 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 5-mm 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 %ΔHDi (mild, −10 to −40 %ΔHDi; severe, −41 to −70 %ΔHDi) at 4 weeks postpuncture. Disc levels with a %ΔHDi 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 × 103 cells per disc in a volume of 10 µl using a 27G hypodermic needle (Hamilton, Reno, NV, http://www.hamiltoncompany.com). For control groups, 10 µl 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 × 103 to 1.5 × 105 cells in a 10 µl 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.siemens.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 = (HDitest/HDiref) × 100%. Percentage change in %HDi (%ΔHDi) was calculated, where %ΔHDi = %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 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 µm 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 × 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 μm/second) was applied, followed by a 30-minute hold period (swelling phase), and then a 10% compressive strain (ramp rate, 0.25 μm/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 μm thickness were adhered to Histobond+ 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 dried samples were sputter-coated with a thin layer of gold (<5 nm) and imaged by scanning electron microscope (SEM) (Leo 1530 FEG, Carl Zeiss, Germany, http://www.zeiss.com) at a magnification of 40 K and an electron high tension voltage of 5 kV [25] to study the nanoscale structure. Morphometric analysis was conducted using a SkyScan CT-Analyser (ver 1.9.3.2) to quantify the diameter of the fibrils and the porosity of the fibril meshwork [26]. Only fibrils lying on the focal plane were taken into account for calculating the porosity, and fibrils lying out of plane were excluded. The porosity of the tissue was expressed as % area not covered by the fibrils. The diameters of 200 fibrils and porosity from 100 images of each group were measured.

Atomic Force Microscopy

Nanoindentation was performed on partially dried NP sections of 8 μm thickness in an atomic force microscope (AFM) (Solver P47-PRO, NT-MDT, Moscow, Russia, http://www.ntmdt.com) using a soft AFM tip (CSG 10, NT-MDT, Moscow, Russia) of nominal force constant 0.1 N/m in contact mode. To indent the samples, the piezo-base was moved up toward the indenter tip by a distance of 100 nm at a rate of 10 nm s−1 (loading phase). The piezo-base was then held for 1 minute (holding phase) and thereafter retracted from the tip at a rate of 2 nm s−1 (unloading phase). We validated that there were no signs of indentation marks by postindentation AFM imaging, indicating that the indentation depth is within the elastic limit. A total of 75 indentation data were recorded on samples of each group in ambient condition. The elastic moduli of the samples were evaluated from the indentation data using equations as described previously [27] (see Supporting Information Methods for more details).

Glycosaminoglycan Quantification by 1,9-Dimethylmethylene Blue Dye Assay

After mechanical testing, the NP specimens were lyophilized and digested overnight at 60°C in 500 µl papain solution (0.3 U/ml papain, 50 mM EDTA, 0.1 M sodium acetate, 5 mM l-cysteine, pH 5.3). Glycosaminoglycan (GAG) content was analyzed by 1,9-dimethylmethylene blue dye (DMMB)-binding assay as previously described [28]. The DNA content of the discs was measured using Hoechst 33258 method [29]. The data were expressed as micrograms of GAG per micrograms of DNA (GAG/DNA).

Histological Analysis and Immunohistochemistry

The endplate-disc-endplate segments were harvested at 12 months post-treatment. The samples were fixed in 4% paraformaldehyde, decalcified at 4°C in Morse's solution (22.5% formic acid and 10% sodium citrate) (Sigma-Aldrich), and embedded in paraffin wax (Paraplast Plus, McCormick Scientific, http://www.fishersci.com). Standard Masson Trichrome staining (using light green dye) was performed on mid-sagittal sections to examine the fibrous collagen content. Multichromatic FAST staining which comprises Alcian blue, safranin O, tartrazine, and fast green dyes was performed as previously described [19] to examine the histological structure as well as the distribution and content of proteoglycan.

To detect protein expression, antigen retrieval was performed on the transverse disc cryosections by incubating in 0.8% hyaluronidase at 37°C for 60 minutes. The sections were washed gently with PBS for 5 minutes then blocked in 0.5% goat serum at room temperature for 40 minutes. The sections were incubated with mouse monoclonal antibodies against collagen I (Abcam, Cambridge, U.K., http://www.abcam.com; Cat. No. ab90395), collagen II (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com; Cat. No. sc-52658) or aggrecan (Abcam; Cat. No. ab3773), or rabbit/goat polyclonal antibodies against CD3 (Abcam; Cat. No. ab828), CD56 (Santa Cruz; Cat. No. sc-1507) in 0.5% goat serum at 4°C overnight. The sections were washed gently with PBS then incubated with Rhodamine Red-conjugated secondary antibodies (Invitrogen) in 0.5% goat serum for 40 minutes. After washing with PBS, the slides were mounted using Vectashield mounting medium with DAPI (Vector Labs, San Francisco, http://www.vectorlabs.com) and visualized at 594 nm for antibody signal and 360 nm for DAPI. For detection of matrix metalloproteinase 12 (MMP-12) expression, the sections were incubated with goat polyclonal antibody against human MMP-12 (Santa Cruz) at 4°C overnight, followed by Vectastain ABC kit (Vector Labs) using fast red for color development.

Analysis of MSC Paracrine Effects

After expansion at passage 5, the human bone marrow-derived MSCs were cultured for 48 hours in DMEM, supplemented with 2% fetal calf serum, 1% penicillin/streptavidin, 1% fungizone, and 1% l-glutamine. The MSC-conditioned media (MSC-CM) was then filtered through a 0.22-µm membrane and stored at −20°C. NP cells were harvested from degenerated human patient disc samples by collagenase and pronase digestion. At passage 2, they were encapsulated in 1.2% alginate solution (Sigma-Aldrich) at 106 cells per ml. The alginate beads were cultured in complete DMEM at 37°C with 5% CO2 for 1 week, followed by culture in MSC-CM or unconditioned control media for 7 days with change of fresh media every 3 days. The beads were digested in 30 U/ml papain solution (Sigma-Aldrich) at 60°C overnight. The GAG content in the lysate was then measured by the DMMB assay and normalized to total protein content measured by the Bradford assays (Bio-Rad, Hercules, CA, http://www.bio-rad.com). For gene expression analysis, the NP cells were released from the alginate beads in dissociation buffer (55 mM sodium citrate, 30 mM disodium EDTA, and 0.15 M sodium chloride, pH 6.8) after 7-day culture in MSC-CM for RNA extraction.

Gene Expression Analysis

Total RNA was extracted from cells using Trizol reagent (Invitrogen), and reverse transcription was performed with a High Capacity RNA to cDNA Kit (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) according to the manufacturer's instructions. Standard TaqMan gene expression assays were performed with StepOnePlus Real-Time PCR System (Applied Biosystems). Genes investigated include aggrecan (AGC1) (Hs00153936_m1), cadherin 2 (CDH2) (Hs00983056_m1), collagen I (COL1A1) (Hs00164004_m1), collagen III (COL3A1) (Hs00943809_m1), fibulin 1 (FBLN1) (Hs0097 2609_m1), fibronectin (FN1) (Hs01549976_m1), HSP47 (Hs00241 844_m1), cytokeratin 18 (KRT18) (Hs01941416_g1), cytokeratin 19 (KRT19) (Hs00761767_s1), matrix metalloproteinase 12 (MMP12) (Hs00899662_m1), matrix Gla protein (MGP) (Hs00179899_m1), RUNX2 (Hs00231692_ml), and SOX9 (Hs00165814_m1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs03929097_g1) was used as an endogenous control. Relative expression levels were determined by the comparative ΔΔCT method.

Statistical Evaluation

For quantitative assessment of T2-weighted MRI signal, disc height index, GAG content, macroscale mechanical test data, and microscale confined compression data, a two-tailed Mann Whitney U-test was performed between experimental groups to calculate p values. Independent samples Kruskal-Wallis test was performed for the nanoindentation data. Independent samples t test was performed for the analysis of the nanoscale tissue porosity and fibril diameter. Paired t test was performed for cell counting. A p value <.05 was considered statistically significant.

Results

Efficacy of MSCs in Inhibiting Disc Degeneration and Fibrogenesis of the Nucleus

The lumbar spine of the rabbit, Oryctolagus cuniculus, bears more relevance to the human spine in terms of anatomy, biomechanics, and loading stimuli than that of other small animal models, such as rodents. We, and others, have demonstrated that physical injury to the disc annulus induces fibrous lesions in the NP core [30, 31]. In puncture-induced lumbar disc degeneration (PDD) in NZW rabbits [19] (Supporting Information Fig. 1a), we can clearly demonstrate progressive degeneration in the affected discs, illustrated by a gradual loss of disc signal in T2-weighted MRI [21] (Supporting Information Fig. 1b, 1d) and a reduction of disc height [20] (Supporting Information Fig. 1c, 1e). Staining with Masson Trichrome revealed fibrous collagen deposition in the NP of these discs (Supporting Information Fig. 1f). A similar fibrotic matrix can be seen in degenerated human specimens (Fig. 1A), suggesting PDD in the rabbit and human disc degeneration involve fibrosis in the disc core.

Figure 1.

Fibrosis of the NP in disc degeneration and its inhibition by MSCs. (A): Masson Trichrome staining of NP samples isolated from nondegenerated (n = 5; ND1–ND5) and degenerated (n = 5; D1–D5) human discs. Samples are arranged according to positivity. Scale bar = 400 μm. (B): Schematic representation of the PDD model and MSC implantation. Lumbar disc degeneration was induced by annulus puncture in NZW rabbits. MSCs were isolated from bone marrow of ChB rabbits and encapsulated in self-assembling peptide hydrogel. The MSC-hydrogel composite was then implanted into the core of degenerated NZW rabbit discs. (C): Long-term evaluation of the effects of MSC dosage on disc degeneration. The effect of MSC on PDD was tested at two different dosages (5 × 103 and 5 × 104 cells) and followed for 12 months. Degeneration status was assessed using D2O-assisted MRI and expressed as percentage loss of HDi relative to the reference level (Δ%HDi). A zero Δ%HDi value indicates no degeneration. *, p ≤ .05; #, p ≤ .057, ^, p ≤ .115 versus phosphate buffered saline (PBS)/hydrogel. (D): Disc height was measured in parallel from spine radiographs to calculate %DHI. *, p ≤ .05; #, p = .057, ^ p ≤ .35 versus PBS/hydrogel. The mortality rate was normal in all groups. Graphs represent mean ± SEM of four pooled independent experiments; Mann-Whitney nonparametric test. (E): Representative photomicrographs of middle disc sections showing compartmental organization, assessed by FAST staining and Masson Trichrome staining at 12 months after implantation (scale bar = 400 μm). Abbreviations: af, annulus fibrosus; ChB, Chinchilla Bastard rabbits; D, degenerated; DHI, disc height index; HDi, H2O:D2O index; mpt, months post-treatment; MSC, mesenchymal stem cell; MRI, magnetic resonance imaging; NZW, New Zealand White; ND, nondegenerated; np, nucleus pulposus; PDD, puncture-induced disc degeneration; PBS, phosphate buffered saline.

To investigate the effect of MSCs on fibrotic events in a rabbit model of PDD, we adopted an interstrain allogeneic scheme (Fig. 1B) using MSCs from outbred rabbits (ChB) to avoid any unknown traits that cause inferior antifibrotic activity due to inbreeding, and because the safety and efficacy of allogeneic MSC therapies have been demonstrated in various clinical trials. Starting with a mild PDD model, we observed inhibition of disc degeneration (Fig. 1C, 1D) when bone marrow-derived MSCs were delivered in encapsulated hydrogels [32] (Supporting Information Fig. 2) at a specific dosage. However, no inhibitory effects were observed in severe PDD models, regardless of MSC dosage (Supporting Information Fig. 3). Consistent with the imaging assessment, MSC-injected discs showed an identifiable NP architecture compared to a normal disc (Fig. 1E). In contrast, discs injected with either PBS or hydrogel alone showed significant fibrous lesions in the NP core as a result of the degenerative process. Quantum dot-labeling of MSCs showed 66.0% engraftment by 4 weeks and 88.8% by 12 weeks after injection without recruitment of T and natural killer (NK) cells (Fig. 2). These results indicate that PDD is associated with NP fibrosis analogous to human disc degeneration and that MSC implantation inhibits both disc degeneration and fibrogenesis in a stage- and dose-specific manner.

Figure 2.

Evaluation of MSC engraftment and activities in vivo. (A): The engraftment of quantum dot-labeled MSCs in the NP during the early phase of degeneration inhibition was determined. MSCs were detected in the NP but not in the AF at 4 wpt with an engraftment rate of 66.0% relative to the initial amount at 0 wpt. The engrafted MSC population increased to 88.8% by 12 wpt, comprising 63.1% of total NP cell population. *, p ≤ .05; #, p = .053; paired t test. Graph represents the mean ± SD of three pooled independent experiments with n = 6 discs. Scale bar = 100 μm. (B): The infiltration of immune cells in the NP (left panel) and AF (right panel) by CD3 and CD56 immunolocalization was assessed after MSC implantation. Rabbit thymus tissue was used as a positive control. There was no evidence for the presence of T lymphocytes and natural killer cells as demonstrated by the negativity for CD3 and CD56, respectively. Scale bar = 200 μm. (C): T2-weighted MRI and disc height evaluation validated that the quantum dot-labeled MSCs exhibited similar inhibitory activity on disc degeneration progression. *, p ≤ .05; #, p ≤ .057. Abbreviations: AF, annulus fibrosus; DAPI, 4',6-diamidino-2-phenylindole; HDi, MRI, magnetic resonance imaging; MSC, mesenchymal stem cell; NP, nucleus pulposus; PBS, phosphate buffered saline; wpt, weeks post-treatment.

Figure 3.

Inhibiting disc fibrosis preserves disc function. (A): Schematic representation of macroscale mechanical test regimen. (B): A typical plot resulting from a bending stiffness test of a motion segment. At the fifth loading cycle, the segment showed different force-displacement relationships at the early (α) and late (β) phases of displacement. (C): Force:displacement ratio at α phase during bending of the motion segments at 12 wpt. Samples from 5-year old rabbits (Aged) were included as control. (D): Similar test at β phase. (E): Axial rotation stiffness was determined by torque of the motion segments at 12 wpt. Samples from 5-year old rabbits (Aged) were included as control. (F): In a confined compression test, the swelling pressure of nucleus pulposus (NP) tissues at 12 wpt was measured as the equilibrium stress reached by the end of the swelling phase. (G): The compressive modulus, K, of the NP tissues at 12 wpt was calculated from the slope of stress versus strain plot. *, p ≤ .05; **, p ≤ .01; *** p ≤ .001; # p = .057; Mann–Whitney nonparametric test. Graphs represent mean ± SD of four pooled independent experiments with n = 8 discs (n = 4 discs for aged rabbit samples). See Supporting Information Table 1 for additional data of mechanical tests. (H): Sagittal MRI of the lumbar motion segments at 12 wpt. A more intensive signal (asterisk) was noted in the MSC-treated discs than in the control discs. (I): Lateral radiographs at 12 wpt with the lower panel showing a higher magnification of the L4/5 levels. There was no aberrant ossification or calcification in the operated discs except mild osteophyte formation (arrow). MSC-treated discs showed wider disc space (asterisk) than control discs. Representative radiograph and MRI images from four independent experiments are shown. Abbreviations: MRI, magnetic resonance imaging; MSC, mesenchymal stem cell; PBS, phosphate buffered saline; wpt, weeks post-treatment.

Spinal Segment Function After MSC Engraftment

Disc degeneration causes first instability of spinal motion segments then an increase in segmental stiffness [33, 34]. Since the presence of fibrotic materials impairs the physiological functions of affected tissues, we interpret changes in segmental kinematics to be a result of disc fibrosis, impinging on its biomechanical function. Therefore, we questioned how NP fibrosis affects spine function. We tested entire disc units [22] (Fig. 3A, 3B) and showed that PDD induces motion stiffness at the lumbar segment to a degree similar to aged animals (5 year-old) (Fig. 3C–3E, also see Supporting Information Table 1). Implantation of MSCs significantly reduced degeneration-induced stiffness. The coexistence of fluid and solid phases is one of the major characteristics of the NP that contributes to its mechanical function in the disc [24]. We found that the injection with MSCs restored the biphasic property of the NP (Fig. 3F, 3G; Supporting Information Table 1), supporting a link between fibrosis reduction and maintenance of NP function in vivo. Annulus ossification or NP calcification may occur in human disc degeneration and can contribute to altered disc mechanics. However, imaging data showed no evidence of these features, either in PDD or MSC-induced reduction of degeneration, while osteophytes were noted at all injury sites (Fig. 3H, 3I). Together, our findings indicate that MSCs ameliorate PDD-induced NP fibrosis and the associated loss of spine function.

Nanoscale Properties of Collagen Fibrils in MSC-Implanted Disc

Collagens are the major players in fibrosis; their participation in fibrogenic processes involves deregulated accumulation and assembly into fibrils of abnormal size. We therefore hypothesized that the link between disc fibrosis and motion segment function is coupled to fibrillogenesis. In situ analysis of the collagen fibrils in the NP of fibrotic discs (PBS and hydrogel injections) showed a significantly larger mean diameter (197.9%, p ≤ 5E–79 versus normal) with a much broader distribution when compared to a normal disc (Fig. 4A, 4B) without apparent changes in the D-periodic band intervals (Supporting Information Fig. 4). Injection of MSCs resulted in reduced fibril diameter (p ≤ 1.02E−17) compared with PBS or hydrogel-injected samples (Fig. 4A, 4B), with more than 48.0% maintenance of fibrils of normal diameter. This observation is consistent with a positive effect of MSCs that prevents the aberrant increase in the matrix porosity observed in fibrotic NP (Fig. 4C). This finding supports a model where collagen molecules in the NP aggregate abnormally into larger fibrils in a fibrotic process, leading to an enlarged interfibrillar space, and MSCs enable better organization and assembly of collagen fibrils in a fibrotic disc for recovery and maintenance of disc function.

Figure 4.

Collagen fibril meshwork formation in disc fibrosis. (A): Assessment of collagen fibrils by scanning electron microscopy (SEM). Collagen fibrils display D-periodic bands. Nanofibrils of the hydrogel carrier (arrowheads) were detected in the nucleus pulposus (NP) treated with either hydrogel or MSC. Scale bar = 1 μm. (B): Frequency and distribution of collagen fibril diameter sampled from SEM images. Vertical dashed lines indicate the mean diameter of fibrils. Asymmetric bimodal distribution was observed in the hydrogel and MSC-treated groups. The percentage of fibrils that fall within two SD from the mean diameter of the normal group (shaded) is indicated. Graphs represent data in mean ± S.D.; *, p ≤ 5E–79 for PBS versus normal; **, p ≤ 1.02E–17 for MSC versus PBS and hydrogel; independent samples t test. (C): Interfibrillar space was evaluated by morphometric analysis to determine the porosity of the matrix in NP. *, p ≤ 3.28E–22; #, p = 1.70E–4; independent samples t test. (D): Frequency and distribution of elastic modulus of collagen fibrils measured by in situ nanoindentation. Q2 indicates the median. The percentage of fibrils that show modulus values below the 25th percentile (Q1) or above the 75th quartile (Q3) of the normal group (shaded) are indicated. * Significant difference versus normal, comparing samples with percentile rank below Q1 or above Q3; Independent-samples Kruskal-Wallis test. (E): Immunodetection of collagen I and II. Increased collagen I deposition was detected in degenerated NP (*) but not in NP implanted with MSCs at 12 wpt. SEM images and graphs represent data of four pooled independent experiments with n = 8 discs, with a total of 200 and 75 fibril samples for diameter and modulus assessments, respectively. A representative immunofluorescence image from two independent experiments is shown for each group (scale bar = 200 μm). Abbreviations: COL1A1, collagen I; COL2A1, collagen II; DAPI, 4',6-diamidino-2-phenylindole; ep, bony endplate; MSC, mesenchymal stem cell; PBS, phosphate buffered saline; wpt, weeks post-treatment.

The extent of intermolecular crosslinking affects the stiffness of collagen fibrils and consequently the overall biomechanical function of tissues. We investigated the mechanical properties of individual fibrils by performing in situ indentation using an AFM [35]. We found that the elastic moduli of NP fibrils below the 25th percentile (Q1) were significantly reduced in the PDD discs (PBS- and hydrogel-injected) compared to normal discs (p ≤ .001) (Fig. 4D). No fibrils in the PDD discs displayed moduli comparable to the Q1 (24.6%) of normal discs. Moreover, the moduli above the 75th percentile (Q3) were significantly elevated in the PDD discs (p ≤ .001). Notably, implantation with MSCs maintained NP fibrils of low modulus (below Q1, p ≥ .317 versus normal), with 10.0% fibrils showing moduli comparable to the Q1 of normal discs. We postulate that the gain in fibril elastic modulus in PDD discs is attributed to collagen I, as abnormally high levels of collagen I are associated with both fibrosis and disc degeneration [36]. Immunostaining for collagen I confirmed increased deposition in the interterritorial matrix of the NP in PDD (PBS and hydrogel injected), but little in MSC-treated discs (Fig. 4E, Supporting Information Fig. 5). No obvious changes in the relative deposition of collagen II, the major collagen type in the disc, were observed (Fig. 4E; Supporting Information Fig. 5).

Figure 5.

Primary action of MSCs on profibrotic mediator activities via paracrine signaling. (A): Quantification of GAG in NP by dimethylmethylene blue assay at 12 wpt. Data were normalized to DNA content quantified by the Hoechst dye assay. Graph represents mean ± SD of three pooled independent experiments with n = 6 discs; **, p ≤ .01; ***, p ≤ .005; Mann–Whitney nonparametric test. (B): Immunohistochemical staining of aggrecan in the central zone of the NP. A representative immunofluorescence image from two independent experiments is shown for each group (scale bar = 200 μm). (C): In situ assessment of GAG content by FAST staining. The central zone of the NP (left panel) and the medial lamellae region of the annulus fibrosus (right panel) are shown. Alcian blue-positive matrix represents highly sulfated GAG. An increase of safranin O positive matrix (arrows) in the NP reflects disc degeneration. A representative microphotograph from three independent experiments is shown for each group (scale bar = 100 μm). (D): Cells isolated from human degenerated NP were embedded in alginate and cultured in bone marrow MSC-CM for 7 days. Gene expression in the NP cells was measured using real-time polymerase chain reaction (PCR). Graph represents mean ± SD of three or four pooled independent experiments; *, p ≤ .05 versus control (nonconditioned medium); Mann–Whitney nonparametric test. (E): Measurement of GAG content in the alginate beads after 7-day culture in MSC-CM. Graph represents mean ± S.D. of three or four pooled independent experiments; N.S. Not significant; Mann-Whitney nonparametric test. (F): Expression of profibrotic marker genes in MSC-CM-treated human NP cells was similarly evaluated by real-time PCR. (G): Immunohistochemical staining of matrix metalloproteinase-12 (MMP-12) in the NP of the puncture-induced disc degeneration model system, with subpanels showing the intracellular and territorial expression of MMP-12 (red) in the hydrogel-treated degeneration control (hydrogel) but not MSC-treated (MSC) samples at higher magnification (scale bar = 100 μm). Abbreviations: AGC1, aggrecan; COL1A1, collagen I; COL3A1, collagen III; CM, conditioned medium; DAPI, 4',6-diamidino-2-phenylindole; HSP47, heat shock protein 47; FN1, fibronectin; FBLN1, fibulin 1; GAG, glycosaminoglycan; MSC, mesenchymal stem cell; MGP, matrix Gla protein; NP, nucleus pulposus; PBS, phosphate buffered saline; wpt, weeks post-treatment.

Modulatory Action of MSCs on Profibrotic Mediators

Based on these findings, we propose that disc degeneration is in part attributed to aberrant polymerization of collagen I fibrils with increased interfibrillar space, altering the mechanical properties and permeability [24], and probably also the microenvironment required for disc cell homeostasis. MSCs provide modulating factors that promote reestablishment of the collagen mesh. To identify potential modulators, we next investigated the proteoglycan content, because the binding of proteoglycans to collagen fibrils is known to inhibit fibril fusion [37] and thereby control fibrosis. Indeed, injection of MSCs resulted in better preservation of the GAG content (Fig. 5A), probably due to elevated aggrecan expression in the MSC-injected disc (Fig. 5B; Supporting Information Fig. 5). Multichromatic proteoglycan staining [19] confirmed the preservation of highly sulfated GAG content (alcian blue positivity) in the MSC-injected discs (Fig. 5C), supporting the hypothesis that MSCs modulate the fibrils through promoting proteoglycan expression in the NP cells.

It is known that MSCs may influence and regulate disc cells via long-range signaling in vivo [38] and in vitro [39, 40], and that extensive intercellular communication via secretory factors is a predominant mechanism of the interaction [40]. Indeed, our cell tracking data indicate a lack of cell-cell contact between MSCs and NP cells. To test whether MSCs secrete factors that promote proteoglycan production directly, we cultured NP cells from patients with degenerated discs in MSC-CM. Expression of KRT19 (cytokeratin 19) (p ≤ .05) and KRT18 (cytokeratin 18), both thought to be markers of NP cells [41], was increased (Fig. 5D), suggesting that MSC-CM actively maintains or promotes the NP phenotype. However, there was no change in the expression of AGC1 (Fig. 5D) or the overall GAG content (Fig. 5E). This suggests that the preservation of proteoglycan in MSC-injected NP in vivo is likely to be a secondary event rather than a primary action of MSCs.

It is possible that MSCs influence proteoglycan levels by ameliorating the degenerative phenotype or by promoting differentiation of the NP progenitor cells [39, 42] into a proteoglycan-producing lineage, such as chondrocytes [38]. However, culturing human NP cells from degenerated discs with MSC-CM had no effect on the expression of the disc degeneration markers FBLN1 and MGP [43] or the chondrogenic markers SOX9 and RUNX2 (Fig. 5D). We then hypothesized that MSCs modulate NP fibrosis via regulation of profibrotic mediators that have been shown to regulate collagen matrix deposition in other tissue fibrosis conditions. Two candidates were MMP12, an elastase that contributes to chronic fibrosis through limiting the expression of extracellular matrix-degrading MMPs [44], and heat shock protein 47 (HSP47), a chaperone for intracellular collagen assembly and processing, known to be induced in the fibrotic liver [45]. We observed significant suppression of MMP12 (p = .049) and HSP47 (p = .02) expression in the MSC-CM treated cells (Fig. 5F). We saw no obvious effects on the expression of COL1A1, COL3A1, and FN1. In the PDD model, we detected less signals in MMP12 immunostaining in the MSC-injected NP in vivo (Fig. 5G; Supporting Information Fig. 6). This suggests MSCs exert a paracrine effect on the NP cells of a degenerating disc that could prevent aberrant fibrillogenesis by regulating specific components of the canonical profibrotic pathway.

Figure 6.

A model of disc degeneration and repair in the context of fibrogenesis. Chronic injurious or inflammatory stimuli induce fibrosis, causing abnormal fibrillogenesis in the NP. Through extended crosslinking and a change in composition of collagen types, NP collagen fibrils gradually increase in size and become more rigid. These aberrant fibrils negatively affect disc mechanics, ultimately leading to compromised motion segment function and abnormal spine kinematics. In addition, the rigid fibrils may inhibit the differentiation or activities of local progenitor cells, eventually limiting remodeling of the extracellular matrix and hence tissue repair. The antifibrotic activities of MSCs can assist the disc to repair through normalizing fibrillogenesis. Abbreviations: HSP, heat shock protein; NP, nucleus pulposus; MMP, matrix metalloproteinase; MSCs, mesenchymal stem cells.

Discussion

Our findings from clinical samples and the two-way in vivo model system of disc degeneration and MSC-mediated intervention provide evidence for our contention that disc degeneration presents features of fibrotic diseases. Through a combination of biochemical, imaging, and biomechanical assays, our study supports a model in which MSCs can assist disc repair by inhibiting the establishment of a fibrotic microenvironment through paracrine signals that may have a potential role in suppressing profibrotic mediator activities (Fig. 6), thereby preserving mechanical function. While the association of MMP12 and HSP47 with human disc degeneration and its fibrosis has yet to be demonstrated, their potential role in fibrotic events is supported by their expression in fibroblasts and/or myofibroblasts in fibrotic conditions of other tissues [46, 47]. Whether resident fibroblasts or other profibrotic effector cells, such as myofibroblasts [4], exist in the degenerated discs and are the targets of regulation merits further investigation. Previous studies indicate that MMP-12 functions to limit the expression of other MMPs such as MMP-2 and MMP-13 that can promote collagen degradation and mediate interleukin (IL)-13-dependent fibrosis [44, 48].

Following repeated injury, tissue undergoes remodeling and fibrosis. The process of fibrosis can be broadly divided into three main overlapping phases: initial triggering, inflammatory cascades, and fibrotic events. In vascularized systems, exaggerated apoptosis of epithelial cells can secrete cytokines which recruit and activate macrophages. The activated macrophages in turn produce fibrogenic factors such as transforming growth factor (TGF)-β1 to promote proliferation and activation of the collagen producing fibroblasts. Macrophages are also essential to mediate the resolution of fibrosis by initiating extracellular matrix degradation [49]. A deregulation of these processes is thought to cause excessive accumulation of matrix, in particular fibrous collagens. Macrophages are also known to produce MMP-12. Interestingly, macrophage-like activities have been recently reported in degenerative human IVD samples [50] and that degenerated IVD from patients with discogenic back pain show upregulated expression of connective tissue growth factor, a downstream effector of TGF-β1 [9]. It is not clear if a deregulation of macrophage activities is also associated with the fibrotic transformation in the disc degeneration process or if MSCs may promote the regression of fibrosis through regulating macrophages. This notion is consistent with the findings that MSCs can secrete cytokines to inhibit macrophage activities through deregulating inflammatory mediators IL-6 and TNF-α [11, 51]. The finding of elevated IL-6 and TNF-α expression in degenerated and herniated IVD also appears to parallel to this hypothesis [52, 53].

Intradiscal implantation studies in various animal models including rodents, rabbits, and beagles indicate that MSCs may promote disc regeneration [16, 54-58]. A preliminary study of two human cases reported that implantation of autogeneic MSCs into the lumbar disc may improve level stability, pain, and other clinical outcomes [59, 60]. Although autogeneic MSC therapy for disc degeneration is preferred, it has limitations in clinical application including a lack of shelf availability and the need to harvest tissues from patients. On the other hand, allogeneic MSC therapy would resolve the issue of shelf availability with the option of implanting MSCs from younger donors that could provide better and longer functional outcomes. Studies in rat or rabbit models have raised the feasibility of allogeneic MSC transplantation [61-64]. Nevertheless, the presumption has not been well validated and the allogeneicity of MSCs could be limited due to the use of inbred strains. By in vivo and ex vivo assessment, we showed that cross-strain transplantation of MSCs alleviate the progression of disc degeneration without inducing a host-versus-graft response, therefore further supporting the use of allogeneic MSCs for treating disc degeneration.

Our cell dosage evaluation indicates that an increase in MSC quantity in the transplantation cannot improve the regenerative capacity but rather negates their effects. Our finding is consistent with previous work that showed a limited nutrition supply in the disc. In this scenario, nutritional competition or overaccumulation of metabolic wastes may have an adverse effect on the activity of MSCs when they are implanted at high quantity. This is also consistent with previous study in a canine model of disc degeneration which showed that the implantation of different quantities of MSCs may lead to varied grades of annulus changes [65]. It is also possible that a high density of MSCs within a confined environment might increase the likelihood of MSC differentiation, which may reduce their native antifibrotic role. Future testing with lower MSC dosages or determining the threshold quantity of MSCs that gives benefits may provide useful information for clinical application.

Our 1-year transplantation study indicated that MSC transplantation delayed rather than arrested disc degeneration in the long term. Previous reports have indicated that MSCs differentiate into a chondrogenic lineage after transplantation into the IVD [38, 56]. It is possible that such differentiation eventually leads to a reduction of their antifibrotic effects. However, it should be noted that the disc height and the NP compartment were still largely maintained during the long-term study, suggesting that the function of the disc might not necessarily be adversely compromised. It is possible that the gradual drop in T2-weighted signal is in part associated with natural ageing [21].

Our data indicate that disc degeneration is associated with fibrosis of the NP, in which the collagen meshwork becomes dominated by fibrils of abnormal size and rigidity. Collagen fibrilogenesis is a complex molecular process co-ordinated by a number of factors such as collagen genetic types, mechanical stress, and aging [66]. In cartilage, collagens II and IX together form the D-periodic, heterotypic thick fibrils (40-nm diameter) and together with collagen XI form the thin (16-nm diameter) fibrils. Collagen types I, III, and V constitute the thick collagen fibrils in mouse decidua. Among the list of collagens that could possibly link to disc degeneration is N-propeptide of collagen I [36]. In our study, the diameter of the collagen fibrils in normal NP is within the range of previously reported values for collagen II fibrils [35, 67]. Furthermore, our data is consistent with a previous study reporting that the collagen fibril diameter appears less uniform in degenerated NP in humans [68]. Our finding of higher porosity in the degenerated NP, pertaining to the thicker and less uniform fibril meshwork, is also in line with the report by Johannessen and Elliott [24] that the permeability of the human disc positively correlates with degeneration. We propose that the correlation to permeability is related to an increase in interfibrillar space owing to the abnormal polymerization of collagen fibrils as a result of disc fibrosis. It is noteworthy that a loss of small leucine-rich proteoglycans (SLRPs) decorin and lumican caused collagen fibril fusion [69, 70] and may accelerate tissue fibrosis [71]. Mice deficient in biglycan were shown to develop signs of disc degeneration [72]. Investigating how MSCs modulate SLRPs may possibly provide additional insights into their antifibrotic activities.

Clinical observations and animal studies indicate that disc degeneration is progressive and irreversible. To date, it is not clear what cells are involved in promoting degeneration progression. Since the acute phase of inflammation is likely to have subsided at the time of MSC implantation (1 month postpuncture), it is assumed that the effects of MSCs on the degeneration progression may be related to chronic inflammatory activities. In the context of the current study and model of tissue fibrosis [73], it is likely to involve macrophage, myofibroblast, and other inflammatory cell activities in response to chronic inflammatory stimuli. Our findings reveal a previously unidentified role of fibrillogenesis in the pathogenesis of disc degeneration. Interestingly, rigid matrix fibrils can transduce unfavorable mechanobiological signals to local progenitors to inhibit their differentiation or activities [74], and reduced progenitor cell activity has been demonstrated in degenerated discs [42]. Recently, we have also found that SLRPs can regulate survival of disc progenitor cells in vitro [75], suggesting a potential link between fibrillogenesis and disc progenitor activities. In this context, we speculate that the unfavorable matrix meshwork generated by fibrosis and the consequent mechanobiological signals may establish a feedback loop that compromises endogenous progenitor cell activities, eventually leading to deregulated self-repair and uncontrolled fibrosis progression that aggravates the hostile microenvironment (Fig. 6). This model may explain the limited amount of tissue remodeling and repair and hence the progressive nature of disc degeneration. The exogenous MSCs may have a distinct function in promoting the repair process through diminishing the feedback loop via their antifibrotic activity.

While we propose that regulators of fibrillogenesis could be an attractive target for activation of self-repair and regression of disc degeneration, our data emphasize the importance of early intervention. Development of sensitive diagnostics, for example, screening of genetic risk factors [76] and sophisticated imaging modalities that can identify predisposed subjects, is therefore highly relevant in future management of the disease.

Conclusion

Our results demonstrated that MSCs can assist IVD repair in part via inhibiting the fibrogenic process. Fibrosis is known as the main pathological feature of common inflammation- or injury-induced organ malfunction as well as systemic autoimmune disorders such as systemic lupus erythematosus, Crohn's disease, and rheumatoid arthritis. Emerging evidence suggests fibrosis accounts for morbidity and mortality in these conditions. Remarkably, accumulating evidence also suggests that local or systemic delivery of MSCs can alleviate a majority of these conditions and promote healing in preclinical settings or even at the bedside. Our study supports a capacity of MSCs in potentiating resident stem cell activities and ultimately tissue repair or regeneration through a regulation on collagen fibrillogenesis, providing mechanistic basis for the MSC-based therapies.

Acknowledgments

We thank Koji Akeda and Yumiko Abe for their guidance in the rabbit spine surgery; Gladys Lo in Department of Diagnostic Radiology, Hong Kong Sanatorium & Hospital for providing the MRI scan service; our colleagues Dino Samartzis for his advice in statistical analysis, and Lai-Ching Li, Stephen Chan and Si-Yun Ng for providing technical assistance. This work is funded by the Area of Excellence grant (AoE/M-04/04) and General Research Fund (HKU7496/05M, HKU7153/06E, and HKU7149/08E) from the Research Grant Council of Hong Kong, and the HKU Foundation Seed Grant (HKUF-DC).

Author Contributions

V.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; D.A.: conception and design of nanoscale/microscale experiments, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; F.L.: collection and/or assembly of data and data analysis and interpretation; V.T., Y.S., R.L. and S.-C.H.: collection and/or assembly of data; A.N., B.T., C.T.L., and K.L.: data analysis and interpretation; E.W.: data analysis and interpretation and administrative support; W.L.: data analysis and interpretation, and financial support; K.M.: data analysis and interpretation and experimental support; D.C.: conception and design, data analysis and interpretation, and final approval of manuscript; K.C.: conception and design, data analysis and interpretation, financial support, and final approval of manuscript. V.Y.L.L. and D.M.K.A. contributed equally to this work.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

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