- Email: [email protected]
A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model
Accepted Manuscript A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model Xiaofeng Chen, Lixin Zhu, Guofeng Wu, Zhihao Liang, Leiluo Yang, Zhicai Du PII:
S1743-9191(16)00153-9
DOI:
10.1016/j.ijsu.2016.02.045
Reference:
IJSU 2591
To appear in:
International Journal of Surgery
Received Date: 12 October 2015 Revised Date:
8 February 2016
Accepted Date: 11 February 2016
Please cite this article as: Chen X, Zhu L, Wu G, Liang Z, Yang L, Du Z, A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model, International Journal of Surgery (2016), doi: 10.1016/ j.ijsu.2016.02.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc
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degeneration in a rabbit model
Xiaofeng Chen, Lixin Zhu*, Guofeng Wu, Zhihao Liang, Leiluo Yang, Zhicai Du
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Department of Orthopedics, Zhujiang Hospital, Southern Medical University,
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Guangzhou, China
*Corresponding author. Department of Orthopedics, Zhujiang Hospital, Southern Medical University, No.253, Gongyedadao Road, Guangzhou 510280, China. Tel:
(Lixin Zhu)
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+86 20 62782578, Fax: +86 20 62782578. Email: [email protected]
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Running title: Comparison of NPSCs and NPCs transplantation in a rabbit model
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A comparison between nucleus pulposus-derived stem cell transplantation and
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nucleus pulposus cell transplantation for the treatment of intervertebral disc
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degeneration in a rabbit model
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Abstract
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Introduction: In recent years, nucleus pulposus cell (NPC) transplantation has been
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used to treat intervertebral disc degeneration (IDD); however, the degenerative nature
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of NPCs influences its effectiveness. Nucleus pulposus-derived stem cells (NPSCs),
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which are self-renewing, have high expansion potential and can adapt to the
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intervertebral disc (IVD) microenvironment and may have a better regenerative
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capacity, which is favourable for treating IDD. The aim of this study was to compare
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the effectiveness of transplantation with NPSCs and NPCs on the regeneration of the
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IVD in rabbit models.
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Methods: NPSCs and NPCs were isolated from human degenerate nucleus pulposus
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tissue by differential adhesion method, and stem cell surface markers were detected
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by flow cytometry. Degenerative discs in rabbits were randomly distributed into three
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groups: NPSCs, NPCs and vehicle control group; the normal discs served as the
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normal control group. Cells of the P3 generation were prepared for transplantation.
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About 20 µl of cell suspension（NPSCs or NPCs）or DMEM was injected into
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corresponding discs, while the normal discs were left untreated. After 8 weeks, disc
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height was evaluated using X-ray, water content was evaluated by MRI, and gene and
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protein expression levels of collagen II and aggrecan in the nucleus were determined
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Results: NPCs and NPSCs from the P3 generation were polygonal and
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spindle-shaped, respectively. Both NPSCs and NPCs strongly expressed surface
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markers CD73, CD90, and CD105 and weakly expressed CD34 and CD45. The
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relative rates of expression of CD73, CD90, and CD105 were higher in NPSCs than in
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NPCs. After 8 weeks, X-ray results showed no significant difference in disc height
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index among the groups (p＞0.05). MRI revealed that the intensity of the nucleus
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pulposus signal was increased in NPSCs (p＜0.05). The results from PCR and ELISA
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demonstrated that NPSCs promoted gene and protein expression of aggrecan instead
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of collagen II (p＜0.05).
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Conclusion: Compared to NPCs, NPSCs harvested by differential adhesion method
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displayed a higher positive rate of stem cell surface markers and showed superior
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regenerative effectiveness for treating IDD in rabbit models. Therefore, NPSCs are
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potential candidates for cell therapy for the regeneration of the IVD.
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Keywords: Intervertebral disc degeneration; Nucleus pulposus-derived stem cells;
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Nucleus pulposus cells; Cell transplantation
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1. Introduction Low back pain is one of the most common health conditions, associated with high
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medical costs and patient morbidity [1]. Intervertebral disc degeneration (IDD) is a
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major cause of low back pain [2]. The normal intervertebral disc (IVD) has three
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distinct components: the central hyperhydrated nucleus pulposus (NP), the outer
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annulus fibrosus, and the upper and lower cartilaginous endplates [3]. Although the
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causes of IDD degeneration are still largely unknown, a decrease in the function and
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number of nucleus pulposus cells (NPCs) is an initial trigger of IDD [4, 5]. Various
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operative methods such as spinal fusion and artificial disc replacement have been used
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to treat degenerative disc diseases and have shown satisfactory results in alleviating
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pain. They are, however, not devoid of complications, including accelerated
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degeneration of the levels adjacent to the fusion or the prosthetic disc’s migration,
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extrusion or failure [6, 7]. Most importantly, these treatments aim to alleviate patients’
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symptoms, but do not target the underlying disease itself. None of these treatments
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aims to restore the biological function of the disc nor slow down or reverse IDD [8, 9].
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Recently, cell transplantation has become one of the major biological procedures used
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to treat IDD [8-10].
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Due to their self-renewing ability, high expansive potential in culture, and
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capacity for multilineage differentiation, stem cells are a more attractive cell source
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for the treatment of IDD [10]. Many types of stem cells have been used in cell
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transplantation for the treatment of IDD, such as bone marrow-derived stem cells
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(BMSCs) [11], adipose-derived stem cells (ADMSCs) [12], human umbilical 3
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cells must survive and function in the harsh IVD microenvironment for successful cell
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therapy. The IVD microenvironment is characterised by high osmolarity, limited
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nutrition, acidic pH, and low oxygen tension [9, 15, 16]. Unfortunately, some studies
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have reported that stem cells lack viability, proliferation, and matrix biosynthesis
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under IVD microenvironment, as it negatively influences the biological and metabolic
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vitality of stem cells and impairs their repair potential [17, 18]. Therefore, it is
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necessary to find other cell sources for cell therapy for IDD.
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Transplantation of NPCs has become one of the major techniques used
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experimentally to prevent IDD. However, NP tissues have low levels of cellular and
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proliferative activity [19]. The regenerative capacity of autologous disc cells
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harvested from herniated discs was questioned in an in vitro study, where the cells lost
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their differentiation potential and ability to synthesize aggrecan and collagen type II
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[20]. Moreover, the application of NPCs is limited because phenotypic changes
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displayed in the degenerated IVD cause NPCs to produce less extracellular matrix
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[21].
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In 2007, Risbud et al [22] provided evidence for the existence of endogenous
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progenitor cells in human NP tissue and demonstrated their capacity for multilineage
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differentiation in vitro. Recently, many researchers have demonstrated the existence
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of NP-derived stem cells (NPSCs) among various species [23, 24] such as rat, mini
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pig, rabbit, and canine. Blanco et al [25] demonstrated that NPSCs isolated from
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human degenerate NP tissue possess a capacity for chondrogenic differentiation 4
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the harsh IVD microenvironment than ADMSCs were. Tao et al [27] harvested
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NPSCs and NPCs through the method of differential adhesion, Tao et al [27]
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harvested NPSCs and NPCs through differential adhesion and found that NPSCs and
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NPCs expanded from adherent primary cells within 1 day and 3 days, respectively.
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In this study, we harvested NPSCs and NPCs from human degenerated nucleus
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pulposus tissue by differential adhesion. First, we aimed to study the differential
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expression of mesenchymal stem cell (MSC) surface markers in NPSCs and NPCs
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and second, to investigate the regenerative effects of NPSCs and NPCs in rabbit
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models of IDD.
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2. Materials and methods
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The research ethical committee of our hospital approved this study, and this
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paper was written in accordance with the Animal Research: Reporting In Vivo
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Experiments (ARRIVE) statement [28].
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2.1. Isolation of NP tissue-derived cells
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All the tissue samples were obtained from patients with moderately degenerative
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lumbar intervertebral disc while undergoing vertebral body fusion. NP tissue was
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detached from surrounding tissues such as vessels, annulus fibrosus, and ligaments.
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Each NP sample was cut into half and assigned for isolation as NPCs or NPSCs.
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Isolation of NPCs was performed by enzymatic digestion. Tissue samples were
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washed three times with PBS and minced into 1-mm3 pieces. Collagenase II solution 5
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for 6 h. The mixture was filtered through a 200-mesh sieve and re-suspended in 10%
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FBS and high-glucose DMEM. Cells suspensions were then seeded in 6-well plates
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and cultured at 37°C in a humidified atmosphere containing 5% CO2. The complete
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medium was changed every 3 days. Cells harvested using this method were labelled
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NPCs. After three passages, NPCs were observed under the inverted microscope, and
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NPCs suspensions were prepared for analysis by flow cytometry and intradiscal
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implantation.
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To isolate NPSCs, differential adhesion was performed as previously reported
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[27]. The cell extraction process was similar to the method used for NPC isolation. NP
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tissue samples were washed with PBS, minced into pieces, and digested with
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collagenase II solution at 37°C for 6 h. The mixture was filtered through a 200-mesh
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sieve and re-suspended in 10% FBS and high-glucose DMEM. Cells suspensions
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were seeded in 6-well plates and cultured at 37°C in a humidified atmosphere
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containing 5% CO2. After 24 h, culture media with non-adherent cells were discarded
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and adherent cells were supplemented with fresh medium. The culture medium was
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renewed every 3 days. After three passages, NPSCs were prepared for identification
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by flow cytometry and for the animal experiments.
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2.2 Flow cytometry assay
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Flow cytometry assay was used to identify the expression of specific surface
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markers in the NPSCs and NPCs. In accordance with the minimal criteria for defining 6
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expression of surface markers like CD73, CD90, CD105, CD34, and CD45 was
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evaluated in this study [29]. About 1 × 106 cells were re-suspended with PBS
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(containing 5% FBS) to produce a single cell solution. NPSCs and NPCs were
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incubated separately with PE-tagged CD73 and CD105, FITC-tagged CD90, and
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APC-tagged CD34 and CD45 at room temperature. Finally, labelled cells were
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washed three times with PBS and surface marker expression was detected using flow
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cytometry.
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2.3. Animal surgery
For induction of IDD, 24 male New Zealand rabbits (weight: approximately 2.5
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kg; age: 6 months) were used. All animals were subjected to surgery and throughout
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the procedures they were housed in cages. The rabbits were monitored daily during
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the entire experimental procedure by an accredited veterinarian, trained in laboratory
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animal science. Lumbar discs 3-6 were designated the target discs for induction of
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degeneration and lumbar discs 6-7 were designated as the normal group. The surgical
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process was conducted as described by Masuda et al [30]. After being anesthetized by
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an intramuscular injection of ketamine and xylidinothiazoline at a 1:1 volume ratio,
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the operative area was shaved using a special razor, and the rabbit was placed in the
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left lateral position. Following sterilization with entoiodine, targeted discs (L3-6)
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were exposed by making a right posterolateral incision, following which the right
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anterolateral annulus fibrosus was punctured with a 21G needle and nucleus pulposus
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After 4 weeks, degenerative discs with a Pfirrmann score of II were selected for the
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intradiscal injection. All the selected discs were randomly assigned to three groups:
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treatment with NPCs, NPSCs, and no cells (vehicle control). About 20 µl of DMEM
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containing NPCs and NPSCs (cell number: 1 × 106) were injected into the assigned
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discs using a 27G needle via the left posterolateral approach, and discs injected with
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DMEM without cells were considered the vehicle control group.
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2.4. Imaging-based evaluation
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One day before and 12 weeks after degeneration induction surgery, lateral
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radiographs of the lumbar discs were performed under general anaesthesia. Two
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diagnostic imaging technologists who were blinded to the study design independently
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assessed the disc height index. Disc height index (DHI) was calculated according to
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following formulas proposed by An et al [31].
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(1)
() DHI=( )( )
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(2)
%DHI= × 100
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DH denotes disc height, LB denotes lower body height, UB denotes upper body
height, and A/M/P denotes anterior/medium/posterior.
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MRI assessment was performed using the 3.0-T imager (German). Rabbits were
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laid in the supine position after being anaesthetised and the T2 weighted sagittal
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images were taken. Two radiologists calculated the relative signal intensity index (RSI)
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of the discs. The signal intensity values for targeted discs were obtained by 8
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quantifying the average values for regions of interest using a CD manager software.
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The RSI was obtained by dividing L2-3 disc signal intensity value (normal disc) by
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that of the targeted discs.
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2.5. PCR assay
Twelve weeks after degeneration was induced, rabbits were euthanized by
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injecting an overdose of pentobarbital intravenously. Samples were collected from the
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sacrificed rabbits, and total RNA was extracted using the TRIzol kit. RNA was
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transcribed into single-stranded cDNA, and the reverse-transcription reaction was
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performed for 15 min at 37°C and then for 5 s at 85°C. Specific primers (Table 1) for
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Col II, AGG, and GADPH were used in this study. The relative levels of gene
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expression of Col II and AGG were normalised to that of GADPH. The relative gene
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expression levels in the NPSCs, NPCs, and normal groups were normalised to those
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in the vehicle control group.
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2.6. Biochemical assay
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Commercial ELISA determined the protein levels of collagen II and aggrecan in
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targeted nucleus pulposus. After rabbits were sacrificed, NP tissues were obtained
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from targeted discs and minced with liquid nitrogen. Lysis Buffer (500µl) was then
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added, which was followed by repeated freezing and thawing. The supernatant was
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collected after centrifugation at 12000 rpm for 30 min. Collected supernatant was
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analysed by ELISA to determine the protein levels and the relative protein content 9
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was normalised to the vehicle control. All samples were tested in duplicate.
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2.7. Statistical analysis Statistical analysis was conducted with SPSS19.0 software. Data were presented
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as mean ± SD. t-tests were performed to determine whether the differences among the
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groups were significant. A p-value of less than 0.05 was considered statistically
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significant.
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3. Results
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No changes in weight or general condition were observed during the
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experimental protocol. No important adverse events were obsered in each group. All
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the animals were used to analysis.
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3.1. Evaluation of isolated cells
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Under inverted microscope, the P3 generation NPCs were polygonal and the P3
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generation NPSCs showed an elongated spindle shape, much like MSCs (Fig. 1A).
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NPSCs and NPCs were identified by flow cytometry, and expression levels of the
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surface markers CD73, CD90, CD105, CD34, and CD45 were tested. Flow cytometry
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results showed that NPSCs were positive for CD73, CD90, and CD105 at rates greater
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than 95%, and negative for CD34 and CD45 (below 5%) (Fig. 2B and 2C). This
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expression pattern is characteristic of MSCs. In the NPCs, expression of CD73, CD90,
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and CD105 was positive but at rates lower than 95% and expression of CD34 and
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CD45 were negative (below 5%).
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3.2. Disc height and MRI assessment X-rays were taken 1 day before and 12 weeks after IDD induction. DHI was
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calculated as previously described. The NPCs, NPSCs, and vehicle control group
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showed no significant difference in %DHI(p ＞ 0.05); however, the value was
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significantly lower than in the normal group (Fig. 2) (p＜0.05). The MRI showed (Fig.
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3) that the signal intensity of targeted discs in the NPSCs group was higher than that
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in all other groups except the normal group. The RSI in the NPSCs group was higher
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than that in the NPCs and vehicle control group (p＜0.05), but lower than that in the
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normal group (p＜0.05).
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3.3. PCR assay
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The level of mRNA expression for the targeted gene is shown in Fig. 4. The
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sequence of the rabbit primers is shown in Fig. 4A. Treatment with NPSCs
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dramatically upregulated mRNA expression of collagen II and aggrecan, and the
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expression level was higher in NPSCs than in NPCs and the vehicle control group but
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lower than in the normal group (p＜0.05).
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3.4. Biochemical assessment
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Expression of collagen II and aggrecan was determined as mentioned above.
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Results showed that the protein expression level of aggrecan in the NPSCs group was
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higher than in the NPCs and vehicle control group, but lower than in the normal group 11
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(p＜0.05). However, collagen II expression showed no significant difference in the
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NPSCs group compared with that in NPCs and the vehicle control group (p＞0.05).
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4. Discussion
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Given the value of cells to the metabolic health of the disc, a reasonable therapy
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is to replace, regenerate, or increase the intervertebral disc cell population [8].
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Autologous disc cells, when implanted in the degenerative discs of dogs, remained
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viable, produced matrix components similar to those in normal discs, and retained
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disc height [8]. Following these findings, investigators injected autologous disc cells
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in human patients after microdiscectomy. The patients demonstrated an increase in
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fluid content and pain relief after 2 years compared to that observed with the control
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group [8]. In another clinical trial, patients who received autologous disc cell
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transplantation showed a lower reduction in fluid content compared with the control
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group and better pain relief after 24 months [32].
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Low cellularity and proliferation of NPCs from human degenerate NP tissue
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limits its clinical application [19]. Recently, NPSCs were identified in NP tissue, even
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in degenerate discs, and displayed preferable proliferation and were able to adapt to
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the harsh IVD microenvironment [25, 26]. Therefore, NPSCs from degenerative discs
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may have considerable potential in the treatment of IDD. In this study, we compared
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the regenerative potential of two types of cells derived from NP tissue for IVD
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regeneration.
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In this study, NPSCs showed a higher positive rate for expression of CD73, 12
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markers CD34 and CD45 was negative in both NPSCs and NPCs (＜5%). Surface
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markers CD73, CD90, and CD105 can be detected in stem cells derived from various
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mesenchymal tissues such as tendon [33], cartilage [34], and endplate [35]. CD34 and
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CD45, which are expressed on the surface of hematopoietic cells [22], are not
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expressed on MSCs. The results showed a significant difference in the expression of
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CD90 and CD105 between NPSCs and NPCs, demonstrating that NP-derived cells
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isolated by differential adhesion method within different days could result in different
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purification rate of progenitors.
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Both NPSCs and NPCs were isolated from human degenerative IVD. Unlike in
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xenogenic cell transplantation in vascular tissues, transplantations in avascular tissue
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such as IVDs do not induce an inflammatory response due to immune privilege [9,
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35]. Therefore, transplantation of xenogenic cells into IVD is feasible. MRI, PCR, and
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biochemical assay results suggest that regenerative effectiveness was, to some extent,
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achieved by injection of NPSCs. However, NPSCs did not display regenerative
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effectiveness with respect to disc height. This can be explained by an insufficient
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increase in glycosaminoglycan content to supplement height loss or because the X-ray
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was insensitive to weeny changes in disc height.
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Both in vitro study and in vivo animal experiments have demonstrated that
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MSCs exert their regenerative effect in three ways: by differentiating into NPC-like
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cells, by activating inherent cells, and via an anti-inflammatory effect [35-37]. In this
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study, NPSCs also displayed regenerative effectiveness in IDD. Except for the 13
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adapt to the adverse IVD microenvironment [27]. Han et al [26] demonstrated that
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NPSCs displayed high levels of viability and proliferation under different pH levels,
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similar to that seen in mild and severe degenerative IVD. In contrast, MRI showed
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that NPC transplantation did not improve NP hydration; this could be attributed to the
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lowered biological character of NPCs [27].
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Being preliminary, this study has some limitations. First, the use of rabbit
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models of IDD cannot fully simulate the process of human IVD degeneration; the use
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of larger animals like rhesus monkeys or sheep as models of IDD may be more
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representative. Second, the duration of this study is insufficient to reflect the complete
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regenerative effect of cell therapy; therefore, future studies should extend the duration
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of the experiment to 24 months. Lastly, the sample size used in this study is relatively
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small; thus, conclusions can be drawn only in relation to this sample.
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As NPSCs showed a better regenerative effect in the animal experiments, it is
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reasonable to conclude that NPSC transplantation may be a superior candidate for
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IDD cell therapy in humans and may overcome the shortcomings of NPCs,
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specifically their senescence and low proliferative ability.
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5. Conclusion
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Compared to NPCs, NPSCs harvested by the differential adhesion method
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displayed a higher positive rate for the expression of stem cell surface markers and
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showed superior regenerative effectiveness for IVD in rabbit models. Therefore, 14
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NPSCs are a potential candidate for cell therapy in the regeneration of IVD.
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10 (2012) 49. [19] J. Mochida, D. Sakai, Y. Nakamura et al., Intervertebral disc repair with activated
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nucleus pulposus cell transplantation: a three-year, prospective clinical study of
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its safety, Eur Cell Mater 29 (2015) 202-212; discussion 212.
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[20] E.S. Vasiliadis, S.G. Pneumaticos, D.S. Evangelopoulos et al., Biologic treatment
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400-409.
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[21] S. Gou, S.C. Oxentenko, J.S. Eldrige et al., Stem cell therapy for intervertebral
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disk regeneration, Am J Phys Med Rehabil 93 (11 Suppl 3) (2014) S122-131.
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[22] M.V. Risbud, A. Guttapalli, T.T. Tsai et al., Evidence for skeletal progenitor cells in the degenerate human intervertebral disc, Spine (Phila Pa 1976) 32 (23) (2007)
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2537-2544.
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[23] H. Henriksson, M. Thornemo, C. Karlsson et al., Identification of cell
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proliferation zones, progenitor cells and a potential stem cell niche in the
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intervertebral disc region: a study in four species, Spine (Phila Pa 1976) 34 (21)
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(2009) 2278-2287.
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cells: implications for regenerative medicine and neural repair, Spine (Phila Pa
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[25] J.F. Blanco, I.F. Graciani, F.M. Sanchez-Guijo et al., Isolation and
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characterization of mesenchymal stromal cells from human degenerated nucleus 17
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pulposus: comparison with bone marrow mesenchymal stromal cells from the
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same subjects, Spine (Phila Pa 1976) 35 (26) (2010) 2259-2265. [26] B. Han, H.C. Wang, H. Li et al., Nucleus pulposus mesenchymal stem cells in
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acidic conditions mimicking degenerative intervertebral discs give better
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performance than adipose tissue-derived mesenchymal stem cells, Cells Tissues
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[27] Tao YQ, Liang CZ, Li H, Zhang YJ, Li FC, Chen G, et al. Potential of co-culture
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of nucleus pulposus mesenchymal stem cells and nucleus pulposus cells in
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hyperosmotic microenvironment for intervertebral disc regeneration. Cell
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biology international. 2013;37:826-34.
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[28] Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving
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research. PLoS Biol. 2010;8:e1000412.
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[29] Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining
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multipotent mesenchymal stromal cells. The International Society for Cellular
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[30]K. Masuda, Y. Aota, C. Muehleman et al., A novel rabbit model of mild,
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reproducible disc degeneration by an anulus needle puncture: correlation between
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[31] H.S. An, K. Takegami, H. Kamada et al., Intradiscal administration of osteogenic
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protein-1 increases intervertebral disc height and proteoglycan content in the 18
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nucleus pulposus in normal adolescent rabbits, Spine (Phila Pa 1976) 30 (1)
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(2005) 25-31; discussion 31-22. [32] H.J. Meisel, T. Ganey, W.C. Hutton et al., Clinical experience in cell-based
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[33] Y. Bi, D. Ehirchiou, T.M. Kilts et al., Identification of tendon stem/progenitor
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[34] S. Alsalameh, R. Amin, T. Gemba et al., Identification of mesenchymal
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progenitor cells in normal and osteoarthritic human articular cartilage, Arthritis
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Rheum 50 (5) (2004) 1522-1532.
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[35] B. Huang, L.T. Liu, C.Q. Li et al., Study to determine the presence of progenitor
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cells in the degenerated human cartilage endplates, Eur Spine J 21 (4) (2012)
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613-622.
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cells in a canine disc degeneration model, J Orthop Res 26 (5) (2008) 589-600.
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[36] A. Hiyama, J. Mochida, T. Iwashina et al., Transplantation of mesenchymal stem
[37] D. Sakai, J. Mochida, T. Iwashina et al., Differentiation of mesenchymal stem
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cells transplanted to a rabbit degenerative disc model: potential and limitations for stem cell therapy in disc regeneration, Spine (Phila Pa 1976) 30 (21) (2005) 2379-2387.
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Figure 1. Cell morphology and flow cytometry analysis. (A) Morphology of NPCs
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(left) and NPSCs (right); (B) Results from flow cytometry; (C) Positive rate of the
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expression of surface markers.
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Figure 2. Disc height evaluation at 12 weeks after IDD induction. (A) X-ray imaging
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results for different groups; (B) Percent disc height index (%DHI) calculated
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according to the X-ray image results.
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Figure 3. MRI assessment at 12 weeks after IDD induction. (A) MRI results; (B)
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relative signal intensity index.
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Figure 4. Level of mRNA expression for collagen II and aggrecan at 12 weeks after
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IDD induction.
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Figure 5. Level of protein expression for collagen II and aggrecan at 12 weeks after
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IDD induction.
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ACCEPTED MANUSCRIPT Highlights: Nucleus pulposus-derived stem cells with higher mesenchymal stem cells surface markers expression can be harvest by differential adhesion method.
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Transplantation of nucleus pulposus-derived stem cells with higher mesenchymal stem cells surface markers showed more favorable regenerative effect for
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intervertebral disc degeneration.
S1743-9191(16)00153-9
DOI:
10.1016/j.ijsu.2016.02.045
Reference:
IJSU 2591
To appear in:
International Journal of Surgery
Received Date: 12 October 2015 Revised Date:
8 February 2016
Accepted Date: 11 February 2016
Please cite this article as: Chen X, Zhu L, Wu G, Liang Z, Yang L, Du Z, A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model, International Journal of Surgery (2016), doi: 10.1016/ j.ijsu.2016.02.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc
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degeneration in a rabbit model
Xiaofeng Chen, Lixin Zhu*, Guofeng Wu, Zhihao Liang, Leiluo Yang, Zhicai Du
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Department of Orthopedics, Zhujiang Hospital, Southern Medical University,
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*Corresponding author. Department of Orthopedics, Zhujiang Hospital, Southern Medical University, No.253, Gongyedadao Road, Guangzhou 510280, China. Tel:
(Lixin Zhu)
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+86 20 62782578, Fax: +86 20 62782578. Email: [email protected]
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Running title: Comparison of NPSCs and NPCs transplantation in a rabbit model
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A comparison between nucleus pulposus-derived stem cell transplantation and
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nucleus pulposus cell transplantation for the treatment of intervertebral disc
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degeneration in a rabbit model
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Abstract
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Introduction: In recent years, nucleus pulposus cell (NPC) transplantation has been
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used to treat intervertebral disc degeneration (IDD); however, the degenerative nature
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of NPCs influences its effectiveness. Nucleus pulposus-derived stem cells (NPSCs),
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which are self-renewing, have high expansion potential and can adapt to the
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intervertebral disc (IVD) microenvironment and may have a better regenerative
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capacity, which is favourable for treating IDD. The aim of this study was to compare
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the effectiveness of transplantation with NPSCs and NPCs on the regeneration of the
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IVD in rabbit models.
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Methods: NPSCs and NPCs were isolated from human degenerate nucleus pulposus
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tissue by differential adhesion method, and stem cell surface markers were detected
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by flow cytometry. Degenerative discs in rabbits were randomly distributed into three
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groups: NPSCs, NPCs and vehicle control group; the normal discs served as the
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normal control group. Cells of the P3 generation were prepared for transplantation.
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About 20 µl of cell suspension（NPSCs or NPCs）or DMEM was injected into
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corresponding discs, while the normal discs were left untreated. After 8 weeks, disc
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height was evaluated using X-ray, water content was evaluated by MRI, and gene and
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protein expression levels of collagen II and aggrecan in the nucleus were determined
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ACCEPTED MANUSCRIPT by real-time PCR and ELISA.
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Results: NPCs and NPSCs from the P3 generation were polygonal and
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spindle-shaped, respectively. Both NPSCs and NPCs strongly expressed surface
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markers CD73, CD90, and CD105 and weakly expressed CD34 and CD45. The
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relative rates of expression of CD73, CD90, and CD105 were higher in NPSCs than in
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NPCs. After 8 weeks, X-ray results showed no significant difference in disc height
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index among the groups (p＞0.05). MRI revealed that the intensity of the nucleus
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pulposus signal was increased in NPSCs (p＜0.05). The results from PCR and ELISA
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demonstrated that NPSCs promoted gene and protein expression of aggrecan instead
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of collagen II (p＜0.05).
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Conclusion: Compared to NPCs, NPSCs harvested by differential adhesion method
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displayed a higher positive rate of stem cell surface markers and showed superior
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regenerative effectiveness for treating IDD in rabbit models. Therefore, NPSCs are
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potential candidates for cell therapy for the regeneration of the IVD.
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Keywords: Intervertebral disc degeneration; Nucleus pulposus-derived stem cells;
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Nucleus pulposus cells; Cell transplantation
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1. Introduction Low back pain is one of the most common health conditions, associated with high
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medical costs and patient morbidity [1]. Intervertebral disc degeneration (IDD) is a
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major cause of low back pain [2]. The normal intervertebral disc (IVD) has three
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distinct components: the central hyperhydrated nucleus pulposus (NP), the outer
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annulus fibrosus, and the upper and lower cartilaginous endplates [3]. Although the
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causes of IDD degeneration are still largely unknown, a decrease in the function and
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number of nucleus pulposus cells (NPCs) is an initial trigger of IDD [4, 5]. Various
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operative methods such as spinal fusion and artificial disc replacement have been used
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to treat degenerative disc diseases and have shown satisfactory results in alleviating
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pain. They are, however, not devoid of complications, including accelerated
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degeneration of the levels adjacent to the fusion or the prosthetic disc’s migration,
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extrusion or failure [6, 7]. Most importantly, these treatments aim to alleviate patients’
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symptoms, but do not target the underlying disease itself. None of these treatments
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aims to restore the biological function of the disc nor slow down or reverse IDD [8, 9].
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Recently, cell transplantation has become one of the major biological procedures used
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to treat IDD [8-10].
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Due to their self-renewing ability, high expansive potential in culture, and
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capacity for multilineage differentiation, stem cells are a more attractive cell source
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for the treatment of IDD [10]. Many types of stem cells have been used in cell
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transplantation for the treatment of IDD, such as bone marrow-derived stem cells
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(BMSCs) [11], adipose-derived stem cells (ADMSCs) [12], human umbilical 3
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cells must survive and function in the harsh IVD microenvironment for successful cell
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therapy. The IVD microenvironment is characterised by high osmolarity, limited
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nutrition, acidic pH, and low oxygen tension [9, 15, 16]. Unfortunately, some studies
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have reported that stem cells lack viability, proliferation, and matrix biosynthesis
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under IVD microenvironment, as it negatively influences the biological and metabolic
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vitality of stem cells and impairs their repair potential [17, 18]. Therefore, it is
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necessary to find other cell sources for cell therapy for IDD.
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Transplantation of NPCs has become one of the major techniques used
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experimentally to prevent IDD. However, NP tissues have low levels of cellular and
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proliferative activity [19]. The regenerative capacity of autologous disc cells
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harvested from herniated discs was questioned in an in vitro study, where the cells lost
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their differentiation potential and ability to synthesize aggrecan and collagen type II
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[20]. Moreover, the application of NPCs is limited because phenotypic changes
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displayed in the degenerated IVD cause NPCs to produce less extracellular matrix
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[21].
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In 2007, Risbud et al [22] provided evidence for the existence of endogenous
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progenitor cells in human NP tissue and demonstrated their capacity for multilineage
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differentiation in vitro. Recently, many researchers have demonstrated the existence
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of NP-derived stem cells (NPSCs) among various species [23, 24] such as rat, mini
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pig, rabbit, and canine. Blanco et al [25] demonstrated that NPSCs isolated from
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human degenerate NP tissue possess a capacity for chondrogenic differentiation 4
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the harsh IVD microenvironment than ADMSCs were. Tao et al [27] harvested
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NPSCs and NPCs through the method of differential adhesion, Tao et al [27]
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harvested NPSCs and NPCs through differential adhesion and found that NPSCs and
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NPCs expanded from adherent primary cells within 1 day and 3 days, respectively.
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In this study, we harvested NPSCs and NPCs from human degenerated nucleus
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pulposus tissue by differential adhesion. First, we aimed to study the differential
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expression of mesenchymal stem cell (MSC) surface markers in NPSCs and NPCs
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and second, to investigate the regenerative effects of NPSCs and NPCs in rabbit
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models of IDD.
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2. Materials and methods
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The research ethical committee of our hospital approved this study, and this
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paper was written in accordance with the Animal Research: Reporting In Vivo
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Experiments (ARRIVE) statement [28].
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2.1. Isolation of NP tissue-derived cells
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All the tissue samples were obtained from patients with moderately degenerative
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lumbar intervertebral disc while undergoing vertebral body fusion. NP tissue was
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detached from surrounding tissues such as vessels, annulus fibrosus, and ligaments.
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Each NP sample was cut into half and assigned for isolation as NPCs or NPSCs.
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Isolation of NPCs was performed by enzymatic digestion. Tissue samples were
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washed three times with PBS and minced into 1-mm3 pieces. Collagenase II solution 5
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for 6 h. The mixture was filtered through a 200-mesh sieve and re-suspended in 10%
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FBS and high-glucose DMEM. Cells suspensions were then seeded in 6-well plates
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and cultured at 37°C in a humidified atmosphere containing 5% CO2. The complete
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medium was changed every 3 days. Cells harvested using this method were labelled
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NPCs. After three passages, NPCs were observed under the inverted microscope, and
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NPCs suspensions were prepared for analysis by flow cytometry and intradiscal
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implantation.
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To isolate NPSCs, differential adhesion was performed as previously reported
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[27]. The cell extraction process was similar to the method used for NPC isolation. NP
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tissue samples were washed with PBS, minced into pieces, and digested with
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collagenase II solution at 37°C for 6 h. The mixture was filtered through a 200-mesh
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sieve and re-suspended in 10% FBS and high-glucose DMEM. Cells suspensions
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were seeded in 6-well plates and cultured at 37°C in a humidified atmosphere
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containing 5% CO2. After 24 h, culture media with non-adherent cells were discarded
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and adherent cells were supplemented with fresh medium. The culture medium was
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renewed every 3 days. After three passages, NPSCs were prepared for identification
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by flow cytometry and for the animal experiments.
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2.2 Flow cytometry assay
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Flow cytometry assay was used to identify the expression of specific surface
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markers in the NPSCs and NPCs. In accordance with the minimal criteria for defining 6
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expression of surface markers like CD73, CD90, CD105, CD34, and CD45 was
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evaluated in this study [29]. About 1 × 106 cells were re-suspended with PBS
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(containing 5% FBS) to produce a single cell solution. NPSCs and NPCs were
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incubated separately with PE-tagged CD73 and CD105, FITC-tagged CD90, and
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APC-tagged CD34 and CD45 at room temperature. Finally, labelled cells were
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washed three times with PBS and surface marker expression was detected using flow
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cytometry.
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2.3. Animal surgery
For induction of IDD, 24 male New Zealand rabbits (weight: approximately 2.5
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kg; age: 6 months) were used. All animals were subjected to surgery and throughout
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the procedures they were housed in cages. The rabbits were monitored daily during
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the entire experimental procedure by an accredited veterinarian, trained in laboratory
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animal science. Lumbar discs 3-6 were designated the target discs for induction of
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degeneration and lumbar discs 6-7 were designated as the normal group. The surgical
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process was conducted as described by Masuda et al [30]. After being anesthetized by
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an intramuscular injection of ketamine and xylidinothiazoline at a 1:1 volume ratio,
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the operative area was shaved using a special razor, and the rabbit was placed in the
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left lateral position. Following sterilization with entoiodine, targeted discs (L3-6)
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were exposed by making a right posterolateral incision, following which the right
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anterolateral annulus fibrosus was punctured with a 21G needle and nucleus pulposus
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After 4 weeks, degenerative discs with a Pfirrmann score of II were selected for the
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intradiscal injection. All the selected discs were randomly assigned to three groups:
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treatment with NPCs, NPSCs, and no cells (vehicle control). About 20 µl of DMEM
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containing NPCs and NPSCs (cell number: 1 × 106) were injected into the assigned
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discs using a 27G needle via the left posterolateral approach, and discs injected with
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DMEM without cells were considered the vehicle control group.
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2.4. Imaging-based evaluation
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One day before and 12 weeks after degeneration induction surgery, lateral
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radiographs of the lumbar discs were performed under general anaesthesia. Two
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diagnostic imaging technologists who were blinded to the study design independently
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assessed the disc height index. Disc height index (DHI) was calculated according to
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following formulas proposed by An et al [31].
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(1)
() DHI=( )( )
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(2)
%DHI= × 100
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DH denotes disc height, LB denotes lower body height, UB denotes upper body
height, and A/M/P denotes anterior/medium/posterior.
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MRI assessment was performed using the 3.0-T imager (German). Rabbits were
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laid in the supine position after being anaesthetised and the T2 weighted sagittal
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images were taken. Two radiologists calculated the relative signal intensity index (RSI)
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of the discs. The signal intensity values for targeted discs were obtained by 8
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quantifying the average values for regions of interest using a CD manager software.
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The RSI was obtained by dividing L2-3 disc signal intensity value (normal disc) by
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that of the targeted discs.
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2.5. PCR assay
Twelve weeks after degeneration was induced, rabbits were euthanized by
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injecting an overdose of pentobarbital intravenously. Samples were collected from the
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sacrificed rabbits, and total RNA was extracted using the TRIzol kit. RNA was
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transcribed into single-stranded cDNA, and the reverse-transcription reaction was
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performed for 15 min at 37°C and then for 5 s at 85°C. Specific primers (Table 1) for
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Col II, AGG, and GADPH were used in this study. The relative levels of gene
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expression of Col II and AGG were normalised to that of GADPH. The relative gene
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expression levels in the NPSCs, NPCs, and normal groups were normalised to those
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in the vehicle control group.
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Commercial ELISA determined the protein levels of collagen II and aggrecan in
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targeted nucleus pulposus. After rabbits were sacrificed, NP tissues were obtained
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from targeted discs and minced with liquid nitrogen. Lysis Buffer (500µl) was then
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added, which was followed by repeated freezing and thawing. The supernatant was
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collected after centrifugation at 12000 rpm for 30 min. Collected supernatant was
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analysed by ELISA to determine the protein levels and the relative protein content 9
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was normalised to the vehicle control. All samples were tested in duplicate.
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2.7. Statistical analysis Statistical analysis was conducted with SPSS19.0 software. Data were presented
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as mean ± SD. t-tests were performed to determine whether the differences among the
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groups were significant. A p-value of less than 0.05 was considered statistically
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significant.
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3. Results
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No changes in weight or general condition were observed during the
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experimental protocol. No important adverse events were obsered in each group. All
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the animals were used to analysis.
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3.1. Evaluation of isolated cells
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Under inverted microscope, the P3 generation NPCs were polygonal and the P3
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generation NPSCs showed an elongated spindle shape, much like MSCs (Fig. 1A).
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NPSCs and NPCs were identified by flow cytometry, and expression levels of the
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surface markers CD73, CD90, CD105, CD34, and CD45 were tested. Flow cytometry
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results showed that NPSCs were positive for CD73, CD90, and CD105 at rates greater
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than 95%, and negative for CD34 and CD45 (below 5%) (Fig. 2B and 2C). This
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expression pattern is characteristic of MSCs. In the NPCs, expression of CD73, CD90,
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and CD105 was positive but at rates lower than 95% and expression of CD34 and
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CD45 were negative (below 5%).
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3.2. Disc height and MRI assessment X-rays were taken 1 day before and 12 weeks after IDD induction. DHI was
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calculated as previously described. The NPCs, NPSCs, and vehicle control group
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showed no significant difference in %DHI(p ＞ 0.05); however, the value was
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significantly lower than in the normal group (Fig. 2) (p＜0.05). The MRI showed (Fig.
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3) that the signal intensity of targeted discs in the NPSCs group was higher than that
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in all other groups except the normal group. The RSI in the NPSCs group was higher
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than that in the NPCs and vehicle control group (p＜0.05), but lower than that in the
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normal group (p＜0.05).
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3.3. PCR assay
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The level of mRNA expression for the targeted gene is shown in Fig. 4. The
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sequence of the rabbit primers is shown in Fig. 4A. Treatment with NPSCs
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dramatically upregulated mRNA expression of collagen II and aggrecan, and the
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expression level was higher in NPSCs than in NPCs and the vehicle control group but
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lower than in the normal group (p＜0.05).
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Expression of collagen II and aggrecan was determined as mentioned above.
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Results showed that the protein expression level of aggrecan in the NPSCs group was
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higher than in the NPCs and vehicle control group, but lower than in the normal group 11
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(p＜0.05). However, collagen II expression showed no significant difference in the
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NPSCs group compared with that in NPCs and the vehicle control group (p＞0.05).
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4. Discussion
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Given the value of cells to the metabolic health of the disc, a reasonable therapy
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is to replace, regenerate, or increase the intervertebral disc cell population [8].
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Autologous disc cells, when implanted in the degenerative discs of dogs, remained
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viable, produced matrix components similar to those in normal discs, and retained
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disc height [8]. Following these findings, investigators injected autologous disc cells
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in human patients after microdiscectomy. The patients demonstrated an increase in
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fluid content and pain relief after 2 years compared to that observed with the control
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group [8]. In another clinical trial, patients who received autologous disc cell
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transplantation showed a lower reduction in fluid content compared with the control
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group and better pain relief after 24 months [32].
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Low cellularity and proliferation of NPCs from human degenerate NP tissue
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limits its clinical application [19]. Recently, NPSCs were identified in NP tissue, even
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in degenerate discs, and displayed preferable proliferation and were able to adapt to
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the harsh IVD microenvironment [25, 26]. Therefore, NPSCs from degenerative discs
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may have considerable potential in the treatment of IDD. In this study, we compared
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the regenerative potential of two types of cells derived from NP tissue for IVD
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regeneration.
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markers CD34 and CD45 was negative in both NPSCs and NPCs (＜5%). Surface
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markers CD73, CD90, and CD105 can be detected in stem cells derived from various
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mesenchymal tissues such as tendon [33], cartilage [34], and endplate [35]. CD34 and
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CD45, which are expressed on the surface of hematopoietic cells [22], are not
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expressed on MSCs. The results showed a significant difference in the expression of
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CD90 and CD105 between NPSCs and NPCs, demonstrating that NP-derived cells
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isolated by differential adhesion method within different days could result in different
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purification rate of progenitors.
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Both NPSCs and NPCs were isolated from human degenerative IVD. Unlike in
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xenogenic cell transplantation in vascular tissues, transplantations in avascular tissue
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such as IVDs do not induce an inflammatory response due to immune privilege [9,
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35]. Therefore, transplantation of xenogenic cells into IVD is feasible. MRI, PCR, and
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biochemical assay results suggest that regenerative effectiveness was, to some extent,
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achieved by injection of NPSCs. However, NPSCs did not display regenerative
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effectiveness with respect to disc height. This can be explained by an insufficient
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increase in glycosaminoglycan content to supplement height loss or because the X-ray
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was insensitive to weeny changes in disc height.
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Both in vitro study and in vivo animal experiments have demonstrated that
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MSCs exert their regenerative effect in three ways: by differentiating into NPC-like
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cells, by activating inherent cells, and via an anti-inflammatory effect [35-37]. In this
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study, NPSCs also displayed regenerative effectiveness in IDD. Except for the 13
ACCEPTED MANUSCRIPT above-mentioned three explanations, another possible reason might be its ability to
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adapt to the adverse IVD microenvironment [27]. Han et al [26] demonstrated that
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NPSCs displayed high levels of viability and proliferation under different pH levels,
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similar to that seen in mild and severe degenerative IVD. In contrast, MRI showed
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that NPC transplantation did not improve NP hydration; this could be attributed to the
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lowered biological character of NPCs [27].
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Being preliminary, this study has some limitations. First, the use of rabbit
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models of IDD cannot fully simulate the process of human IVD degeneration; the use
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of larger animals like rhesus monkeys or sheep as models of IDD may be more
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representative. Second, the duration of this study is insufficient to reflect the complete
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regenerative effect of cell therapy; therefore, future studies should extend the duration
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of the experiment to 24 months. Lastly, the sample size used in this study is relatively
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small; thus, conclusions can be drawn only in relation to this sample.
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As NPSCs showed a better regenerative effect in the animal experiments, it is
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reasonable to conclude that NPSC transplantation may be a superior candidate for
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IDD cell therapy in humans and may overcome the shortcomings of NPCs,
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specifically their senescence and low proliferative ability.
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5. Conclusion
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Compared to NPCs, NPSCs harvested by the differential adhesion method
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displayed a higher positive rate for the expression of stem cell surface markers and
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showed superior regenerative effectiveness for IVD in rabbit models. Therefore, 14
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NPSCs are a potential candidate for cell therapy in the regeneration of IVD.
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Figure 1. Cell morphology and flow cytometry analysis. (A) Morphology of NPCs
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(left) and NPSCs (right); (B) Results from flow cytometry; (C) Positive rate of the
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expression of surface markers.
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Figure 2. Disc height evaluation at 12 weeks after IDD induction. (A) X-ray imaging
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results for different groups; (B) Percent disc height index (%DHI) calculated
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according to the X-ray image results.
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Figure 3. MRI assessment at 12 weeks after IDD induction. (A) MRI results; (B)
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relative signal intensity index.
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Figure 4. Level of mRNA expression for collagen II and aggrecan at 12 weeks after
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IDD induction.
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Figure 5. Level of protein expression for collagen II and aggrecan at 12 weeks after
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IDD induction.
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ACCEPTED MANUSCRIPT Highlights: Nucleus pulposus-derived stem cells with higher mesenchymal stem cells surface markers expression can be harvest by differential adhesion method.
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Transplantation of nucleus pulposus-derived stem cells with higher mesenchymal stem cells surface markers showed more favorable regenerative effect for
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intervertebral disc degeneration.