Co-culturing nucleus pulposus mesenchymal stem cells with notochordal cell-rich nucleus pulposus explants attenuates tumor necrosis factor-α-induced senescence
Li et al. Stem Cell Research & Therapy
Co-culturing nucleus pulposus mesenchymal stem cells with notochordal cell-rich nucleus pulposus explants attenuates tumor necrosis factor-?-induced senescence
Xiao-Chuan Li 0
Mao-Sheng Wang 0
Wei Liu 0
Cheng-Fan Zhong 0
Gui-Bin Deng 0
Shao-Jian Luo 0
Chun-Ming Huang 0
0 Department of Orthopaedic Surgery, Gaozhou People's Hospital , Guangdong 525200 , China
Background: Cell therapy for the treatment of intervertebral disc degeneration (IDD) faces serious barriers since tissue-specific adult cells such as nucleus pulposus cells (NPCs) have limited proliferative ability and poor regenerative potential; in addition, it is difficult for exogenous adult stem cells to survive the harsh environment of the degenerated intervertebral disc. Endogenous repair by nucleus pulposus mesenchymal stem cells (NPMSCs) has recently shown promising regenerative potential for the treatment of IDD. Notochordal cells (NCs) and NC-conditioned medium (NCCM) have been proven to possess regenerative ability for the treatment of IDD, but this approach is limited by the isolation and passaging of NCs. Our previous study demonstrated that modified notochordal cellrich nucleus pulposus (NC-rich NP) has potential for the repair of IDD. However, whether this can protect NPMSCs during IDD has not been evaluated. Methods: In the current study, tumor necrosis factor (TNF)-? was used to mimic the inflammatory environment of IDD. Human NPMSCs were cocultured with NC-rich NP explants from healthy rabbit lumbar spine with or without TNF-?. Cell proliferation and senescence were analyzed to investigate the effect of NC-rich NP explants on TNF-?-treated NPMSCs. The expression of mRNA encoding proteins related to matrix macromolecules (such as aggrecan, Sox-9, collagen I?, and collagen II?), markers related to the nucleus pulposus cell phenotype (including CA12, FOXF1, PAX1, and HIF-1?), and senescence markers (such as p16, p21, and p53), senescence-associated proinflammatory cytokines (IL-6), and extracellular proteases (MMP-13, ADAMTS-5) was assessed. The protein expression of CA12 and collagen II was also evaluated. Results: After a 7-day treatment, the NC-rich NP explant was found to enhance cell proliferation, decrease cellular senescence, promote glycosaminoglycan (GAG), collagen II, and CA12 production, upregulate the expression of extracellular matrix (ECM)-related genes (collagen I, collagen II, SOX9, and ACAN), and enhance the expression of nucleus pulposus cell (NPC) markers (HIF-1?, FOXF1, PAX1, and CA12). Conclusion: Modified NC-rich NP explants can attenuate TNF-?-induced degeneration and senescence of NPMSCs in vitro. Our findings provide new insights into the therapeutic potential of NC-rich NP for the treatment of IDD.
Intervertebral disc degeneration; Notochordal cell-rich nucleus pulposus explants; Nucleus pulposus mesenchymal stem cells; Senescence; TNF-?
Intervertebral disc degeneration (IDD) is the
pathological basis of lower back pain, and current treatment
options are mainly aimed at relieving pain symptoms;
however, these fail to repair the degenerated intervertebral
disc (IVD) [
]. The main characteristics of age-related
IDD are a decrease in resident cells and a decline in
extracellular matrix (ECM) deposition [
]. Therefore, it has
been suggested that resident cells play an important role
in maintaining nucleus pulposus (NP) homeostasis by
sustaining cell numbers and synthesizing new matrix [
During IDD, the number of resident cells decreases;
therefore, restoring the degenerating disc using healthy cells
would represent a logical treatment strategy.
Over the past decades, several reports have proven
that mesenchymal stem cells (MSCs) can differentiate
into nucleus pulposus-like cells [
nucleus pulposus cells (NPCs), used as tissue-specific adult
cells, were reported by many studies for treating IDD.
However, NPCs have limited proliferative ability and
poor regenerative potential; moreover, exogenous adult
stem cells cannot survive in the degenerated IVD
environment due to the associated harsh conditions such as
strong forces, acidic pH, hypoxia, hyperosmolarity, and
limited nutrients . Hence, studying endogenous repair
might provide us with a new method for regenerative
therapy to treat IDD.
Endogenous tissue repair, which relies on the
regenerative ability of tissue-specific stem cells (SCs), has been
verified in human liver, skin, muscle, nervous system,
heart, and bone [
]. To date, studies on the
regenerative potential of endogenous repair mechanisms have
only begun to shed light on IDD in many animal models
and in humans. Risbud et al. were the first to
demonstrate the existence of progenitor cells in the NP ;
subsequently, several studies have proposed the
existence of SCs in mouse, rabbit, porcine, canine, bovine,
and rhesus monkey NP tissue [
nucleus pulposus mesenchymal stem cells (NPMSCs) have
shown promising regenerative potential for IDD .
Unfortunately, several recent studies have reported
decreases in cell numbers and the deterioration of NPMSC
properties during IDD [
]. Based on this, a
promising strategy would stimulate native NPMSC proliferation
and differentiation during this condition.
An increasing number of studies has demonstrated
that disc degeneration is mainly associated with
]. Inflammatory cytokines can significantly
promote cell degeneration, whereas cell degeneration is
also related to increases in inflammatory cytokines [
Tumor necrosis factor (TNF)-?, a typical inflammatory
cytokine, can increase cell senescence, autophagy,
apoptosis, and proliferation [
]. Many in-vitro studies have
reported that TNF-? triggers a range of pathogenic responses
in the disk cells and also affects disc vitality in vivo via
transfer through the endplate [
]. This suggests that
TNF-? plays a critical role in IDD. It is important to
understand the process of NP degeneration to uncover an
effective strategy to repair the damage associated with this
condition. Based on these facts, we deduced that inhibiting
inflammatory cytokine-induced disc degeneration might be
a possible strategy for the prevention and treatment
of this condition.
To date, notochordal cells (NCs) have been found in
the embryonic notochord structure of chordates [
human NCs disappear at approximately age 10, whereas
the degeneration of the IVD is initiated shortly after this
]. Subsequently, NCs were considered to hold
potential for the regeneration of IDD by improving NPC
viability and the expression of matrix proteins [
Several recent studies have suggested the robust
regenerative potential of NCs or notochordal cell-conditioned
medium (NCCM) for the treatment of IDD, and that this
probably occurs through the stimulation of resident
NPCs via secretory factors [
23, 26, 27
]. However, it is
very difficult to promote the proliferation of NCs in vitro
because these cells are sensitive to nutrient deprivation
in this environment. In addition, NCCM can lose some
trophic factors during production and storage processes.
Hence, novel modifications based on the regenerative
potential of NCs are urgently needed to deal with these
shortages. Our previous research demonstrated that
modified NC-rich NP explants can improve cell
proliferation, cell viability, and NC phenotype [
whether this improvement can maintain the regenerative
function of NCs has not been investigated. In this study,
the biological potential of NC-rich NP in the protection
of TNF-?-treated NPMSCs was tested. Moreover,
endogenous repair of IDD might provide an additional
means to obtain sufficient ECM and cells to achieve a
healthy NP. Therefore, the aim of this study was to
investigate the effects of NC-rich NP explant coculture on
cell morphological features, proliferation, senescence,
and extracellular gene/protein expression in NPMSCs.
Human NP tissue was harvested from four male patients
with a diagnosis of lumbar disc herniation and where
the degeneration degree was Pfirrmann grade II. The age
range was 37?52 years, with a mean age of 45.5 years.
Informed consent was obtained from each patient. In
addition, 36 3-month-old male New Zealand white
rabbits (weight 1500?2000 g) were used in this study. The
experiment was performed according to the amended
declaration of Helsinki and was approved by the
Committee of Gaozhou People?s Hospital (no. 2016?007).
Harvesting rabbit NCs, the NC-rich NP explant model, and sample grouping of samples
NC-rich NP was harvested according to our previous
]. Briefly, after intravenous general anesthesia
(3% pentobarbital sodium, 1 ml/kg), the rabbits were
sacrificed and the NC-rich NP tissue from L4/5, L5/6, or L6/7
was harvested and washed with Dulbecco?s modified Eagle?s
medium (DMEM) containing 100 U/ml
penicillin/streptomycin. Subsequently, NP tissue was partially digested with
0.1% collagenase II (Sigma-Aldrich) for 2 h. Then, all
samples were placed in 24-well transwell inserts in triplicate
and incubated in culture medium at 37 ?C under 5% CO2.
The medium was removed and replaced every second day.
NCs were isolated from rabbit NP tissue and digested
with 0.2% collagenase II (Sigma-Aldrich) for 6 h. Cells
were then cultured in culture medium at 37 ?C under
5% CO2. To determine whether NC-rich NP explants
can inhibit TNF-?-induced NPMSC senescence, cells
were cultured in low-serum medium (DMEM/F-12
supplemented with 1% fetal bovine serum (FBS)). A diagram
of the schematic is shown in Fig. 1. After culture in
serum-free medium for 24 h, samples were divided into
three groups as follows: group 1 was treated with
NC-rich-NP as the control, group 2 was treated with
10 ng/ml TNF-?, and group 3 was treated with 10 ng/ml
TNF-? and NC-rich NP explant as the coculture group.
Isolation of human NPMSCs
NPMSCs were isolated and harvested as previously
]. Briefly, NP samples were collected and
immediately transported to a cell culture room under
sterile conditions. After the annulus fibrosus and cartilaginous
endplates were removed and washed at least three times,
the NP tissues were mechanically minced into small pieces
(< 1 mm3) and digested with 0.2% collagenase II (Sigma,
USA) for 4 h at 37 ?C in a humidified incubator. The
suspended cells were then filtered with a 200-?m mesh filter
and centrifuged at 200 ? g for 5 min, which was followed
by two washes with phosphate-buffered saline (PBS).
Finally, the cell pellets were cultured as an explant in
standard MSC expansion medium, consisting of low-glucose
DMEM (HyClone), 10% fetal calf serum (Gibco), and 1%
penicillin/streptomycin (Gibco) in 25-cm2 cell culture
flasks at a density of 1 ? 105 cells/ml; cells were cultured
in a humidified incubator at 37 ?C under 5% CO2.
After 24 h, the suspended cells and medium were
removed, and the adherent cells were cultured and
expanded by completely replacing the medium every
2?3 days. As the cells reached 70?80% confluency, the
primary cells were harvested and passaged. Passage 1
(P1) NPMSCs were harvested with 0.25%
trypsin-ethylenediaminetetraacetic acid (EDTA; Sigma) for 1 min and
subcultured at a ratio of 1:3. After the cells were gradually
passaged, P3 cells were harvested for identification and
cryopreserved for experiments (Fig. 2a).
Cell viability assay for NC-rich NP explant model
To assess NC viability in the NC-rich NP explant model
after culturing for 7 days, NC-rich NP explants were
dyed with fluorogenic ester calcein-AM (CAM; Dojindo)
to detect live cells, and with propidium iodide (PI;
Sigma-Aldrich) to detect dead cells. The tissues were
incubated with 2 mM CAM and 4.5 mM PI for 30 min
at 37 ?C in the dark and gently washed with PBS three
times. A fluorescence microscope (CFM-300; Nikon)
was used for image acquisition.
Senescence-associated ?-galactosidase (SA-?-gal) staining
After 7 days of incubation, NPMSCs were analyzed using
a Senescence ?-Galactosidase Staining Kit (Beyotime
Institute of Biotechnology). Briefly, cells were washed with
PBS, fixed in the SA-?-gal fixative solution for 15 min at
room temperature, rinsed three times with PBS, and
then incubated in SA-?-gal working solution (Reagents
A, B, C, and X-Gal) overnight at 37 ?C under
atmospheric conditions. Quantification was performed by
counting the number of SA-?-gal-positive cells and the
total number of cells from three randomly selected areas
for each sample.
Cells were harvested and the cell pellet was resuspended
in PBS to 106 cells/100 ?l. The cell suspension was
incubated with the following antibodies: HLA-DR-APC,
CD34-FITC, CD45-PE, CD73-PE, CD90-FITC, and
CD105-FITC (eBioscience). An isotype control antibody
(eBioscience) was used for each. Approximately 5 ?l of
each antibody (1:10 dilution, according to the antibody
datasheets) per tube was added, and this was incubated
for 15 min in the dark at 2?8 ?C. Cells were completely
washed with PBS and resuspended in 1% (w/v)
paraformaldehyde (Sangon Biotech, China). Samples were
subjected to flow cytometry (BD Biosciences, USA) and the
data were analyzed by FlowJo software (FlowJo LLC,
Ashland, OR, USA).
Trilineage differentiation assay
We tested the multidifferentiation potential of NPMSCs
toward osteogenic, adipogenic, and chondrogenic lineages
in vitro as previously described [
]. Briefly, osteogenic
differentiation of MSCs was induced with 10% (v/v) FBS,
1 mM ?-glycerol phosphate, 10?8 M dexamethasone, and
50 ?g/ml ascorbic acid (Cyagen, China). Adipogenic
differentiation was induced with 10% (v/v) FBS, 500 ?M
1-methyl-3-isobutylxanthine, 10?9 M dexamethasone, and
60 ?M indomethacin (Cyagen). Chondrogenic
differentiation was induced in cell and micromass culture in the
presence of 10 ng/ml transforming growth factor
(TGF)-?3, 50 ?g/ml ascorbic acid, 1% (v/v)
insulin-transferrin-selenium (ITS) solution, 10?7 M dexamethasone,
and 100 ?M sodium pyruvate (Cyagen).
Induction was stopped after 3 weeks and alizarin red
staining, oil red O staining, and toluidine blue and alcian
blue staining were performed to assess osteogenic,
adipogenic, and chondrogenic differentiation, respectively.
2.7.1 Alizarin red staining
To identify mineral deposits by alizarin red staining,
cells in culture medium were fixed with 70% ethanol for
10 min and stained with 0.5% alizarin red (pH 4.1) for
2.7.2 Oil red O staining
To localize lipid droplets, cell layers were fixed with
4% paraformaldehyde for 30 min and incubated in oil
red O solution for 15 min. Finally, the cultures were
extensively washed with water to remove excess stain and
then microscopically observed.
2.7.3 Alcian blue staining or toluidine blue staining
To detect chondrogenic differentiation, cells were
rinsed three times with PBS, fixed with 4%
paraformaldehyde for 15 min at room temperature, washed with
PBS, and stained with 1% alcian blue for 15 min. Finally,
the cultures were extensively washed with water to
remove excess stain and then microscopically observed.
Moreover, to visualize the deposition of sulfated
glycosaminoglycans (GAGs), slides were incubated with 1%
alcian blue or 1% toluidine blue in 0.1 M HCl overnight.
Finally, cultures were washed extensively with distilled
water and photographed.
Cell proliferation assay
To measure cell proliferation, a Cell Counting Kit-8
(CCK-8; Dojindo Laboratories, Japan) was used as
previously described [
]. Briefly, NPMSCs were seeded in
24-well plates (1 ? 104 cells/well) and different groups
were incubated for 1, 3, 5, and 7 days. After removing
the culture medium and NC-rich NP explant, 10 ?l
CCK-8 solution was added to 100 ?l fresh medium and
the mixture was incubated at 37 ?C for 1 h. Finally, the
samples were added to 96-well plates for final
measurements. Absorbance was measured at 450 nm using a
microplate absorbance reader (Bio-Rad, USA). A blank
96-well plate was used for the zero setting. In addition,
cell numbers in 24-well plates (1 ? 104 cells/well) were
also calculated after culturing for 1, 3, 5, and 7 days. All
experiments were performed three times for every sample.
First-generation NPMSCs were plated in flat-bottomed
24-well plates (1 ? 104/well) and fixed with 4%
paraformaldehyde, permeabilized with 0.2% triton X-100 in PBS
(PBS-T) for 10 min, blocked with PBS containing 5%
FBS, and incubated with antibodies against collagen II
and carbonic anhydrase 12 (CA12) (1:100, Abcam, UK)
at 4 ?C overnight. As a negative control, cells were
incubated with isotype IgG control antibodies under similar
conditions. After the cells were washed, they were
incubated with anti-rabbit secondary antibody (Jackson,
USA) at a dilution of 1:100 for 1 h at room temperature.
Following this, cell nuclei were stained with DAPI
solution (1:1000; Invitrogen) for 5 min at room temperature.
The samples were examined and photographed using a
fluorescence microscope (FV-1000; Olympus). For
quantitative examination, the immunostaining results for the
animal specimens were analyzed using Image-Pro Plus
software (Version 5.1, Media Cybernetics, Inc. USA).
Western blot assay of type II collagen and CA12
Protein extracts from NPMSCs were prepared in 2 ?
SDS lysis buffer containing phosphatase and proteinase
inhibitors. Protein extracts were subjected to 6% or 12%
SDS-PAGE. Protein was separated and then transferred
to a polyvinylidene fluoride membranes by
electroblotting. The membrane was rinsed in water and blocked
with freshly prepared PBS containing nonfat dry milk
(5%) for 60 min at room temperature with constant
agitation. The membrane was then incubated with
human type II collagen (1:5000, Abcam, UK) and CA12
(1:1000, Abcam, UK) antibodies overnight at 4 ?C with
agitation. After washing the membrane three times with
PBS-T, a secondary goat anti-mouse horseradish
peroxidase (HRP)-conjugated antibody was added and
incubated at room temperature for 2 h. Following another
three washes with PBS-T, protein bands were visualized
using the LiCoR Odyssey imager (LI-COR Biosciences,
Lincoln, NE, USA), and semiquantification was
performed using the LiCoR Odyssey imager software.
Biochemical evaluation of GAG/DNA
The glycosaminoglycan (GAG) content was determined
by performing a dimethylmethylene blue (DMMB) assay
]. Briefly, after the NPMSCs were washed with PBS
three times to eliminate the influence of NP explant
tissue or culture medium, NPMSCs were incubated in
solution containing 1 mg/ml papain in 100 mM sodium
phosphate (pH 6.5), 5 mM L-cysteine, and 5 mM EDTA
overnight at 60 ?C. The GAG content in NPMSCs was
determined by the reaction with DMMB, and optical
density was measured at 525 nm using a microplate
]. GAG concentrations were calculated
using a standard curve obtained from chondroitin sulfate
standards (Sigma). The DNA content of the samples was
measured with bisbenzimidol fluorescent dye (Hoechst
33,258; Sigma). The standard curve was obtained with
known concentrations of calf thymus DNA. The amount
of DNA in each sample was calculated based on the
Real-time polymerase chain reaction (RT-PCR) analysis
The expression of genes encoding markers of senescence
(p16, p21, and p53), senescence-associated
proinflammatory cytokines (interleukin (IL)-6), extracellular proteases
(MMP13, ADAMTS-5), a notochordal cell marker
(brachyury), NPC markers (FOXF1, Pax-1, CA12, and
HIF-1?), ECM molecules (collagen I?1, collagen II?1,
Sox-9, and aggrecan), and markers for the three lineages
(LPL, PPAR2, ALP, Runx2) was analyzed by RT-PCR.
The primers used in this study are shown in Table 1
(Sangon Biotech Corporation). Briefly, after NPMSCs
were incubated under different conditions for 7 days,
1 ?g RNA, extracted using TriPure Isolation Reagent
(Roche, Switzerland), was reverse-transcribed into cDNA
using a First Strand cDNA Synthesis Kit (Roche) as per
the manufacturer?s instructions. Then, a reaction
mixture containing cDNA, SYBR Green Mix (Toyobo,
Japan), and primers (Table 1) was subjected to RT-PCR
(CFX96 Real-Time System, Bio-Rad) according to the
manufacturer?s recommended conditions. ?-actin was
used as an internal reference, and relative gene
expression was determined using the 2???Ct method.
The mean values obtained were compared by analysis of
variance (ANOVA) with SPSS13.0 software (SPSS). Data
are presented as mean ? standard deviation values. A
Student?s t test or one-way ANOVA was used for
comparisons between groups; differences were considered
statistically significant at P values < 0.05.
Identification of human NPMSCs
Cells isolated from human degenerated IVDs displayed
fibroblast-like or spindle-shaped morphology in
monolayer culture, indicating plastic adhesion ability (Fig. 2a).
Based on immune phenotypic assays, cells were positive
for CD73, CD90, and CD105 expression (> 95%, Fig. 2b)
and negative for CD34, CD45, and HLA-DR (< 2%,
Fig. 2b). In multilineage differentiation tests, all cells were
capable of osteogenic, chondrogenic, and adipogenic
differentiation in the respective types of differentiation
induction media after 3 weeks (Fig. 2c?j). Moreover, the
expression level of osteogenic, chondrogenic, and
adipogenic differentiation markers were significantly increased
in all induction groups compared with those in the control
groups (Fig. 2k?m). In summary, the obtained cells met
the ISCT criteria for NPMSCs, including adhesive
characteristics, SC phenotypic expression, and multilineage
NC morphology, phenotype, and viability in NC-rich NP explant model
After 7 days of culture, the NCs cultured in ECM were
found to be agminated in several small cluster structures
(Fig. 3a). Cell morphologies were round or polygonal
with different sized vacuoles in the cytoplasm (Fig. 3a).
For cell viability, CAM/PI staining was used, and green
staining indicated viability whereas dead cells were
stained red (Fig. 3a). NCs from L4/5, L5/6, and L6/7
segments exhibited similar live cell percentages, with 94 ?
5% viable cells in the L4/5 segment, 92 ? 7% in the L5/6,
and 98 ? 2% in the L6/7 (Fig. 3b). In addition, the
expression levels of the NC markers brachyury and keratin
18 (KRT18) were also similar, with no significant
differences among the three groups after 7 days of culture,
whereas the expression of both genes was significantly
higher than that in NCs (Fig. 3c, d).
NC-rich NP explants decrease cell senescence in TNF-?
In terms of cell morphology, NPMSCs in the control
group showed compact parallel or vortex populations,
whereas the TNF-?-treated cells exhibited disorderly
distribution after 7 days of culture (Fig. 4a). In addition,
NPMSCs in the control group had elongated spindle
shapes, whereas TNF-?-treated cells had many slender
processes (Fig. 4a). However, this senescent cell
morphology was somewhat attenuated upon coculture with
NC-rich NP explants (Fig. 4a). Furthermore, cell
senescence was analyzed by SA-?-gal staining (Fig. 4a). An
increased number of SA-?-gal-positive NPMSCs was
observed with TNF-? treatment compared with that in
the control group after 7 days (P < 0.05; Fig. 4b). However,
when TNF-?-treated cells were cocultured with NC-rich
NP explants, a reduction in SA-?-gal-positive cells was
observed, and this difference was significant (P < 0.05;
Fig. 4b). For cell senescence-related gene analysis, TNF-?
significantly upregulated the expression of p16, p21, and
p53 compared with that in the control group. However,
NC-rich NP explants decreased the expression of these
markers after TNF-? treatment (P < 0.05; Fig. 4c?e).
Finally, the expression of senescence-associated
proinflammatory cytokines (IL-6) and extracellular proteases
(MMP-13, ADAMTS-5) was tested, and TNF-? was found
to increase the expression of all three genes compared
with that in the control group; in contrast coculturing
with NC-rich NP explants led to a decline in these
markers (P < 0.05; Fig. 4f?h). Taken together, these results
indicate that NC-rich NP explants can attenuate
TNF-?-induced NPMSC senescence.
NC-rich NP explants promote the proliferation of TNF-?
Cell growth curves were used to evaluate the proliferative
ability of NPMSCs after performing CCK-8 assays. The
optical density (OD) value of TNF-?-treated NPMSCs was
significantly lower than that of the control group on days 1,
3, 5, and 7 (P < 0.05; Fig. 5). However, coculture with
NC-rich NP explants increased the proliferation of
TNF-?-treated NPMSCs, as indicated by markedly higher
OD values on days 5 and 7 (P < 0.05; Fig. 5a). Additionally,
significantly lower numbers of NPMSCs were detected in
the coculture group compared with that in the
TNF-?-treated group on days 5 and 7 (both P < 0.05;
Fig. 5b), while in both TNF-?-treated NPMSCs, a higher
cell number was observed in the NC-rich NP coculture
group, with a significant difference at day 7 (P < 0.05;
Fig. 5b). These results suggested that NC-rich NP can
attenuate the inhibitory effects of TNF-? on NPMSC
NC-rich NP explants enhance the expression of NP cell
markers in TNF-?-treated NPMSCs
CA12 is known as a specific marker of NP cells; here, we
examined the expression of this marker in TNF-?-treated
NPMSCs with or without coculture with NC-rich NP
explants. As shown in Fig. 6a, b, the expression of CA12
was downregulated in TNF-?-treated cells compared with
that in the control condition; however, it was upregulated
upon coculture with NC-rich NP explants (P < 0.05).
Western blot analysis also showed that the level of CA12
was decreased in the TNF-?-treated condition but was
increased with NC-rich NP explant coculture (P < 0.05;
Fig. 6c, d). A similar trend was found based on RT-PCR
analysis of the NP cell markers CA12, Forkhead box F1
(FOXF1), and paired box 1 (PAX1) (P < 0.05; Fig. 6e, g, h).
Furthermore, hypoxia inducible factor (HIF)-1? is
continuously expressed in NP cells and plays a pivotal role in
NP biology by precisely regulating essential cellular
functions such as differentiation and survival [
]. In this
study, NC-rich NP explants significantly inhibited
TNF-?-mediated downregulation of HIF-1? levels
compared with the expression in untreated groups, indicating
that the application of NC-rich NP explants can
significantly promote differentiation into nucleus pulposus-like
cells (P < 0.05; Fig. 6f ).
NC-rich NP explants increase ECM-related gene
expression and GAG/DNA in TNF-?-treated NPMSCs
To evaluate the effect of NC-rich NP on the biological
synthesis of ECM, immunofluorescence for collagen II
and the GAG/DNA ratio was analyzed in NPMSCs. The
collagen II optical density and the GAG/DNA ratio
significantly decreased in the TNF-?-treated group
(Fig. 7a?c). However, this was found to be markedly
inhibited in the coculture group (Fig. 7a?c). Western
blot analysis also displayed the same trend, where
collagen II was decreased in TNF-?-treated medium and
increased in the coculture group (Fig. 7d, e). Finally, we
also investigated matrix synthesis by evaluating the
expression of genes encoding collagen type I?1, collagen
type II?1, aggrecan, and Sox-9 (Fig. 7f?i). Although
matrix molecule (aggrecan, Sox-9, and collagen II?1)
expression decreased in TNF-?-treated NPMSCs, NC-rich
NP significantly abolished this inhibitory effect (Fig. 7f?h).
Conversely, collagen type I?1 expression was shown to
decrease further in the coculture group compared with
that in the TNF-?-treated group (Fig. 7i). Collectively,
these results indicate that NC-rich NP can promote
matrix synthesis in TNF-?-treated NPMSCs.
NCs and NCCM have been reported to possess
regenerative potential for the treatment of IDD [
whether this ability can also be effective for endogenous
NPMSCs remains unclear. In addition, although NCs or
the application of NCCM has been associated with
numerous effects during the repair of IDD, the degeneration
of the native NP tissue environment and the loss of some
therapeutic factors during production and
cryopreservation have limited further study [
]. Therefore, a modified
NC-rich NP explant culture model involving partial
digestion of NP tissue was used in our previous study [
improved NC-rich NP culture model demonstrated
promising repair potential for human NPCs by prolonging cell
viability and maintaining native environments. Based on
that, the effect of coculturing NPMSCs with NC-rich NP
explants with TNF-? treatment was investigated in the
present study. These inflammatory conditions might
indicate a more compelling effect and accordingly provide
additional incentive to produce sufficient ECM and
resident cells during IDD.
To date, although a number of studies have shown the
activating effects of NCs for the treatment of IDD, the
regeneration potential was mild or invalid due to the
disruption of the physiological organization of NCs [
]. In this study, cell viability and the expression of
NC phenotype-related genes after NC-rich NP explant
were tested in rabbit L4/5, L5/6, and L6/7 IVDs; the
results demonstrated no significant differences,
indicating that the NC-rich NP explant model was comparable
among the three segments used in this study and that
these could be used randomly. Hence, a series of
experiments was performed in the current study using the
NC-rich NP?NPMSC coculture system. We found that
NC-rich NP explants could stimulate the anabolism of
degenerative NPMSCs by enhancing cell proliferation,
decreasing cellular senescence, and promoting GAG,
collagen II, and CA12 production, together with
upregulating the expression of ECM-related genes (collagen I,
collagen II, SOX9, and ACAN) and enhancing the
phenotype of NPCs (HIF-1?, FOXF1, PAX1, and CA12
expression). Collectively, our results demonstrate that
NC-rich NP explants can potentially promote a
nondegenerated NP phenotype. Our results are in accordance
with some similar studies on the regenerative potential
of NCs; specifically, these cells were thought to produce
nutritional components or secrete growth factors [
]. Importantly, factors secreted by NCs isolated from
different species can have cross-species regenerative
effects on human degenerative cells .
Inflammation is the main pathological process
associated with disc degeneration, and TNF-?, as a typical
inflammatory cytokine, plays a crucial role in inflammatory
cytokine-induced disc degeneration [
]. In this study,
TNF-? was added to the culture medium to induce a
degenerative environment similar to IDD, thereby
simplifying the complex inflammatory milieu. This differed
considerably from the results of numerous previous studies
regarding physiological conditions, further suggesting the
beneficial potential of NC-rich NP explants on NPMSCs
for the treatment of IDD. In the present study, TNF-?
successfully induced premature senescence and
detrimentally affected properties such as cell morphology, cell
proliferation, cell senescence, and the expression of matrix
macromolecules in NPMSCs. Subsequently, NC-rich NP
explants were shown to exhibit an anti-inflammatory
effect during IDD by inhibiting these adverse outcomes,
similar to that observed for NPCs used in our previous
]. Therefore, the therapeutic function of
NC-rich NP explants represents a promising strategy for
preventing and treating IDD.
NPMSCs are considered to play a crucial role in the
maintenance of normal NP tissue homeostasis [
However, the gradual increase in NPMSC senescence during
IDD has a detrimental effect, and inhibiting this
senescence of NPMSCs is thus considered an important
strategy for the treatment of IDD [
]. Our results
suggest that NC-rich NP explants can attenuate the
premature senescence of NPMSCs in an inflammatory
microenvironment. The telomere-based p53-p21-pRb and the
stress-based p16-pRb pathways are predominant pathways
in IDD [
]. Our results suggest that both pathways are
involved in the effect of TNF-? on NPMSCs in vitro, and
the extrinsic factor p16 was shown to play a crucial role as
demonstrated by its marked upregulation. Moreover, this
study also demonstrates that coculture with NC-rich NP
can result in the downregulation of senescence-associated
proinflammatory cytokines (IL-6) and extracellular
proteases (MMP-13, ADAMTS-5), which further strengthens
During the progression of IDD, NPMSC numbers
decrease quickly [
15, 45, 46
]. Therefore, regeneration
therapy including promoting cell proliferation is a promising
and feasible option. In accordance with the positive effects
of NCCM and NCs [
], NPMSC proliferation decreased
in the TNF-?-treated group, whereas proliferation was
restored upon NC-rich NP?NPMSC coculture. Furthermore,
analysis indicated that the increase in cell number was due
to cell proliferation rather than the inhibition of cell death.
These data are of great importance for developing potential
treatments aimed at inhibiting the reduction in cell number
that is observed during IDD.
To evaluate phenotypic alterations in NPMSCs, we
also examined the expression of NP cell markers
following different treatments. It has been shown that TNF-?
can downregulate the expression NP markers, leading to
altered expression of NP-associated markers such as
CA12, FOXF1, PAX1, and HIF-1?. However, the
application of NC-rich NP explants had a significant protective
effect on the expression of HIF-1?, FOXF1, PAX1, and
CA12. It has been noted that FOXF1, PAX1, and CA12
are highly expressed in NP cells and that the expression of
HIF-1? is closely related to cell function in low-oxygen
environments. Our results suggest that NC-rich NP
explants can promote NPMSC differentiation into the NP
Apart from the above, the decline in ECM is also a key
issue. The balance between ECM anabolism and
catabolism by disc cells is disturbed by proinflammatory
cytokines during IDD [
]. Hence, inhibition of these
inflammatory cytokine-mediated pathological processes
might promote NPMSC differentiation and result in
increased deposition of ECM. Our study proved that
NC-rich NP explants can increase matrix synthesis by
promoting gene expression of aggrecan and collagen II?1
and the protein synthesis of collagen II and GAG. In
addition, the expression of Sox-9, indicative of a healthy
NP phenotype, was higher in the coculture group than in
the TNF-?-treated group. This finding further indicates
that NC-rich NP explants have the potential to stimulate
degenerative NPMSCs to differentiate into NP-like cells.
In addition, the reduction in collagen I?1 expression
suggests that the inhibitory effect of NC-rich NP during
fibrosis further facilitates NPMSC differentiation. Taken
together, these results indicate that NC-rich NP explants
can promote matrix synthesis in TNF-?-treated NPMSCs.
This study also has some limitations. Although TNF-?
can mimic the inflammatory effects of IDD to some
extent, the actual environment associated with this
condition is complex, with various factors that are difficult to
completely recapitulate. Furthermore, although the
conclusion was based on in-vitro evidence, there is no
precise animal model to examine the therapeutic effects on
disc degeneration in vivo. Although the positive effect of
NC-rich NP on degenerated NPMSCs during IDD
regeneration was shown, the exact mechanism is still
unclear and requires further study. The next step would be
to determine this mechanism and to confirm this in vivo
using animal models of IDD.
In conclusion, our study reveals that modified NC-rich
NP explants have a positive effect on NPMSCs in an
anti-inflammatory microenvironment by increasing cell
proliferation, decreasing cellular senescence, and
promoting ECM accumulation. Therefore, our results
provide novel insights into the therapeutic potential of
NC-rich NP for the treatment of IDD and might supply
additional motivation to produce sufficient ECM and
resident cells for the treatment of IDD.
ANOVA: Analysis of variance; CA12: Carbonic anhydrase 12; CAM: Calcein-AM;
DMEM: Dulbecco?s modified Eagle?s medium; DMMB: Dimethylmethylene
blue; ECM: Extracellular matrix; EDTA: Ethylenediaminetetraacetic acid;
FBS: Fetal bovine serum; FOXF1: Forkhead box F1; GAG: Glycosaminoglycan;
HIF: Hypoxia inducible factor; IDD: Intervertebral disc degeneration;
IL: Interleukin; IVD: Intervertebral disc; KRT18: Keratin 18; MSC: Mesenchymal
stem cell; NCCM: Notochordal cell-conditioned medium; NC: Notochordal
cell; NP: Nucleus pulposus; NPC: Nucleus pulposus cell; NPMSC: nucleus
pulposus mesenchymal stem cell; OD: Optical density; P: Passage; PAX1: Paired
box 1; PBS: Phosphate-buffered saline; PI: Propidium iodide; RT-PCR: Real-time
polymerase chain reaction; SA-?-gal: Senescence-associated ?-galactosidase;
SC: Stem cell; TNF: Tumor necrosis factor
The authors would like to thank Min Xiao, Southern Medical University, for
her invaluable help and advice in the revision of this study.
This work was supported by grants from the Guangdong Provincial Key
Laboratory of Orthopedics and Traumatology, Orthopedic Research Institute.
Availability of data and materials
The details of information used and analyzed for the current study are
available from the corresponding author on reasonable request.
L-XC, W-MS, and H-CM were responsible for the overall design of the study
and drafted the final manuscript. L-XC, Z-CF, and L-SJ carried out all the
experiments. D-GB made critical revisions to the final draft. L-XC and L-W
acquired all the data and analyzed the data. All authors read and approved
the final manuscript.
Ethics approval and consent to participate
The study was approved by the governing animal research ethics committee in
Gaozhou People?s Hospital, Guangdong, China (no. 2016-007). Ethical approval
for the human research was given by the ethics committee of Gaozhou
People?s Hospital, Guangdong, China (no. 2016-007). Informed consent was
obtained from all study subjects.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
1. Li XC , Tang Y , Wu JH , et al. Characteristics and potentials of stem cells derived from human degenerated nucleus pulposus: potential for regeneration of the intervertebral disc . BMC Musculoskelet Disord . 2017 ; 18 ( 1 ): 242 .
2. Sakai D , Grad S. Advancing the cellular and molecular therapy for intervertebral disc disease . Adv Drug Deliv Rev . 2015 ; 84 : 159 - 71 .
3. Li XC , Wu YH , Bai XD , et al. BMP7 -based functionalized self-assembling peptides protect nucleus pulposus-derived stem cells from apoptosis in vitro . Tissue Eng Part A . 2016 ; 22 ( 19 -20): 1218 - 28 .
4. Arkesteijn IT , Smolders LA , Spillekom S , et al. Effect of coculturing canine notochordal, nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration . Arthritis Res Ther . 2015 ; 17 : 60 .
5. Potier E , de Vries S, van Doeselaar M , Ito K. Potential application of notochordal cells for intervertebral disc regeneration: an in vitro assessment . Eur Cell Mater . 2014 ; 28 : 68 - 81 .
6. Huang YC , Leung VY , Lu WW , Luk KD . The effects of microenvironment in mesenchymal stem cell-based regeneration of intervertebral disc . Spine J . 2013 ; 13 : 352 - 62 .
7. Li Z , Peroglio M , Alini M , Grad S. Potential and limitations of intervertebral disc endogenous repair . Curr Stem Cell Res Ther . 2015 ; 10 ( 4 ): 329 - 38 .
8. Grad S , Peroglio M , Li Z , Alini M. Endogenous cell homing for intervertebral disk regeneration . J Am Acad Orthop Surg . 2015 ; 23 ( 4 ): 264 - 6 .
9. Risbud MV , Guttapalli A , Tsai TT , et al. Evidence for skeletal progenitor cells in the degenerate human intervertebral disc . Spine . 2007 ; 32 : 2537 - 44 .
10. Liu S , Liang H , Lee S-M , et al. Isolation and identification of stem cells from degenerated human intervertebral discs and their migration characteristics . Acta Biochim Biophys Sin Shanghai . 2017 ; 49 ( 2 ): 101 - 9 .
11. Blanco JF , Graciani IF , Sanchez-Guijo FM , et al. Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus: comparison with bone marrow mesenchymal stromal cells from the same subjects . Spine . 2010 ; 35 : 2259 - 65 .
12. Sang C , Cao X , Chen F , et al. Differential characterization of two kinds of stem cells isolated from rabbit nucleus pulposus and annulus fibrosus . Stem Cells Int . 2016 ; 2016 : 8283257 .
13. Navaro Y , Bleich-Kimelman N , Hazanov L , et al. Matrix stiffness determines the fate of nucleus pulposus-derived stem cells . Biomaterials . 2015 ; 49 : 68 - 76 .
14. Shim EK , Lee JS , Kim DE , et al. Autogenous mesenchymal stem cells from the vertebral body enhance intervertebral disc regeneration by paracrine interaction: an in vitro pilot study . Cell Transplant . 2016 ; 25 ( 10 ): 1819 - 32 .
15. Sakai D , Nakamura Y , Nakai T , et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc . Nat Commun . 2012 ; 3 : 1264 .
16. Zhao Y , Jia Z , Huang S , et al. Age-related changes in nucleus pulposus mesenchymal stem cells: an in vitro study in rats . Stem Cells Int . 2017 ; 2017 : 6761572 .
17. Miguelez-Rivera L , Perez-Castrillo S , Gonzalez-Fernandez ML , et al. Immunomodulation of mesenchymal stem cells in discogenic pain . Spine J . 2017 ; 18 ( 2 ): 330 - 42 .
18. Sun Z , Yin Z , Liu C , et al. IL -1beta promotes ADAMTS enzyme-mediated aggrecan degradation through NF-kappaB in human intervertebral disc . J Orthop Surg Res . 2015 ; 10 : 159 .
19. Li P , Gan Y , Xu Y , et al. 17beta-estradiol attenuates TNF-alpha-induced premature senescence of nucleus pulposus cells through regulating the ROS/NF-kappaB pathway . Int J Biol Sci . 2017 ; 13 ( 2 ): 145 - 56 .
20. Markova DZ , Kepler CK , Addya S , et al. An organ culture system to model early degenerative changes of the intervertebral disc II: profiling global gene expression changes . Arthritis Res Ther . 2013 ; 15 ( 5 ): R121 .
21. Stemple DL . Structure and function of the notochord: an essential organ for chordate development . Development . 2005 ; 132 ( 11 ): 2503 - 12 .
22. Chen J , Yan W , Setton LA . Molecular phenotypes of notochordal cells purified from immature nucleus pulposus . Eur Spine J . 2006 ; 15 ( Suppl 3 ): S303 - 11 .
23. Erwin WM , Islam D , Inman RD , et al. Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration . Arthritis Res Ther . 2011 ; 13 ( 6 ): R215 .
24. Gantenbein B , Calandriello E , Wuertz-Kozak K , et al. Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia . BMC Musculoskelet Disord . 2014 ; 15 : 422 .
25. de Vries SA , van Doeselaar M , Meij BP , et al. The stimulatory effect of notochordal cell-conditioned medium in a nucleus pulposus explant culture . Tissue Eng Part A . 2016 ; 22 ( 1-2 ): 103 - 10 .
26. Korecki CL , Taboas JM , Tuan RS , Iatridis JC . Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype . Stem Cell Res Ther . 2010 ; 1 ( 2 ): 18 .
27. Purmessur D , Schek RM , Abbott RD , et al. Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells . Arthritis Res Ther . 2011 ; 13 ( 3 ): R81 .
28. Bai XD , Li XC , Chen JH , et al. Coculture with partial digestion: notochordal cell-rich nucleus pulposus tissue activates degenerative human nucleus pulposus cells . Tissue Eng Part A . 2017 ; 23 ( 15 -16): 837 - 46 .
29. Sun Z , Luo B , Liu Z , et al. Effect of perfluorotributylamine-enriched alginate on nucleus pulposus cell: implications for intervertebral disc regeneration . Biomaterials . 2016 ; 82 : 34 - 47 .
30. Farndale RW , Sayers CA , Barrett AJ . A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures . Connect Tissue Res . 1982 ; 9 ( 4 ): 247 - 8 .
31. Farndale RW , Buttle DJ , Barrett AJ . Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue . Biochim Biophys Acta . 1986 ; 883 ( 2 ): 173 - 7 .
32. Templeton DM . The basis and applicability of the dimethylmethylene blue binding assay for sulfated glycosaminoglycans . Connect Tissue Res . 1988 ; 17 ( 1 ): 23 - 32 .
33. Choi H , Merceron C , Mangiavini L , et al. Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling . Autophagy . 2016 ; 12 : 1631 - 46 .
34. Tao YQ , Liang CZ , Li H , et al. Potential of co-culture of nucleus pulposus mesenchymal stem cells and nucleus pulposus cells in hyperosmotic microenvironment for intervertebral disc regeneration . Cell Biol Int . 2013 ; 37 ( 8 ): 826 - 34 .
35. Dai J , Wang H , Liu G , et al. Dynamic compression and co-culture with nucleus pulposus cells promotes proliferation and differentiation of adipose-derived mesenchymal stem cells . J Biomech . 2014 ; 47 ( 5 ): 966 - 72 .
36. Strassburg S , Richardson SM , Freemont AJ , Hoyland JA. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype . Regen Med . 2010 ; 5 ( 5 ): 701 - 11 .
37. Potier E , Ito K. Can notochordal cells promote bone marrow stromal cell potential for nucleus pulposus enrichment? A simplified in vitro system . Tissue Eng Part A . 2014 ; 20 ( 23 -24): 3241 - 51 .
38. Wang F , Gao ZX , Cai F , et al. Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation . Oncotarget . 2017 ; 8 ( 34 ): 57800 - 12 .
39. Kwon WK , Moon HJ , Kwon TH , et al. Influence of rabbit notochordal cells on symptomatic intervertebral disc degeneration: anti-angiogenic capacity on human endothelial cell proliferation under hypoxia . Osteoarthr Cartil . 2017 ; 25 ( 10 ): 1738 - 46 .
40. Le Maitre CL , Hoyland JA , Freemont AJ . Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile . Arthritis Res Ther . 2007 ; 9 ( 4 ): R77 .
41. Shi R , Wang F , Hong X , et al. The presence of stem cells in potential stem cell niches of the intervertebral disc region: an in vitro study on rats . Eur Spine J . 2015 ; 24 ( 11 ): 2411 - 24 .
42. Liu J , Tao H , Shen C , et al. Biological behavior of human nucleus pulposus mesenchymal stem cells in response to changes in the acidic environment during intervertebral disc degeneration . Stem Cells Dev . 2017 ; 26 ( 12 ): 901 - 11 .
43. Marfia G , Navone SE , Di Vito C , et al. Gene expression profile analysis of human mesenchymal stem cells from herniated and degenerated intervertebral discs reveals different expression of osteopontin . Stem Cells Dev . 2015 ; 24 ( 3 ): 320 - 8 .
44. Wang F , Cai F , Shi R , et al. Aging and age-related stresses: a senescence mechanism of intervertebral disc degeneration . Osteoarthr Cartil . 2016 ; 24 ( 3 ): 398 - 408 .
45. Mizrahi O , Sheyn D , Tawackoli W , et al. Nucleus pulposus degeneration alters properties of resident progenitor cells . Spine J . 2013 ; 13 ( 7 ): 803 - 14 .
46. Molinos M , Cunha C , Almeida CR , et al. Age-correlated phenotypic alterations in cells isolated from human degenerated intervertebral discs with contained hernias . Spine . 2018 ; 43 ( 5 ): E274 - 84 .
47. Sakai D , Andersson GB . Stem cell therapy for intervertebral disc regeneration: obstacles and solutions . Nat Rev Rheumatol . 2015 ; 11 ( 4 ): 243 - 56 .
48. Feng C , He J , Zhang Y , et al. Collagen-derived N-acetylated proline-glycineproline upregulates the expression of pro-inflammatory cytokines and extracellular matrix proteases in nucleus pulposus cells via the NF-kappaB and MAPK signaling pathways . Int J Mol Med . 2017 ; 40 ( 1 ): 164 - 74 .