Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells
Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells
He Liu 0 1
Jianxun Ding 0 1
Jincheng Wang 0 1
Yinan Wang 0 1
Modi Yang 0 1
Yanbo Zhang 0 1
Fei Chang 0 1
Xuesi Chen 0 1
0 1 Department of Orthopedics, The Second Hospital of Jilin University , Changchun , P. R. China , 2 Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun , P. R. China , 3 Academy of Translational Medicine, The First Bethune Hospital of Jilin University , Changchun , P. R. China
1 Academic Editor: Xing-Ming Shi, Georgia Regents University , UNITED STATES
The persistent inflammation of rheumatoid arthritis (RA) always leads to partial synovial hyperplasia and the destruction of articular cartilage. Bone marrow mesenchymal stem cells (BMMSCs) have been proven to possess immunosuppressive effects, and widely explored in the treatment of autoimmune diseases. However, poor inhibitory effect on local inflammatory state and limited capacity of preventing destruction of articular cartilage by systemic BMMSCs transplantation were observed. Herein, toward the classical type II collagen-induced arthritis in rats, the combination treatment of microfracture and in situ transplantation of thermogel-encapsulated BMMSCs was verified to obviously down-regulate the ratio of CD4+ to CD8+ T lymphocytes in peripheral blood. In addition, it resulted in the decreased levels of inflammatory cytokines, such as interleukin-1, tumor necrosis factor- and anticollagen type II antibody, in the serum. Simultaneously, the combination therapy also could inhibit the proliferation of antigen specific lymphocytes and local joint inflammatory condition, and prevent the articular cartilage damage. The results indicated that the treatment programs could effectively stimulate the endogenous and exogenous BMMSCs to exhibit the immunosuppression and cartilage protection capability. This study provided a new therapeutic strategy for autoimmune inflammatory diseases, such as RA.
Funding: This work was financially supported by the
National Natural Science Foundation of China (No.
81171681 and 51303174), the National Science
Foundation for Post-doctoral Scientists of China (No.
2013M530990), and the Jilin Provincial Research
Foundation for Basic Research, China (No.
201215055). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Rheumatoid Arthritis (RA) is an autoimmune disease, which mainly behaves as symmetry
peripheral joint chronic inflammation, joint swelling and pain, articular cartilage damage and
Competing Interests: The authors have declared
that no competing interests exist.
even deformity, and eventually leads to dysfunction . The current therapeutic procedures, that
is, mainly through oral administration of various drugs, focus on controlling inflammation and/
or delaying or even preventing the progression of disease . The symptoms of RA patients can
be ameliorated partly, while the therapeutic effect on individuals still remains undesirable .
Therefore, so far, there is no effective disease-targeting treatment for RA patients, who have to
bear sustaining pain and a risk of cartilage damage, and have the final fate of arthroplasty .
Mesenchymal stem cells (MSCs) are widely distributed in various tissues, including bone
marrow, fat, synovial membrane, perichondrium, periosteum, et al. . Among them, bone
marrow mesenchymal stem cells (BMMSCs) have attracted extensive attention for their
fascinating ability of differentiation into sundry cells, such as osteoblasts, chondrocytes and
adipocytes, which have been applied in diversified tissue engineering . As a typical example, our
previous study confirmed that the full-thickness cartilage defect could be effectively repaired
through the intra-articular admission of uncultured BMMSCs , and the local cell therapy for
cartilage defects of RA seem to be a reasonable scheme. What must be considered is that RA
patients are confronted with the risk of cartilage re-injury. In addition, synovial fluid obtained
from RA patients shows significantly reduced BMMSCs recruitment compared to normal
donors, which is an important origin of cartilage restoration . Therefore, both inhibiting local
joint inflammation and preventing cartilage destruction synchronously are rather tough.
Interestingly, BMMSCs also exhibit powerful function on regulating immune response,
including suppressing the proliferation of T lymphocytes and inhibiting inflammatory mediators
. These properties give BMMSCs the opportunity to restrict various autoimmune diseases,
such as RA, multiple sclerosis, systemic lupus erythematosus, crohn's disease, and so on [10
12]. Autologous BMMSCs transplantation sounds perfect for the treatment of RA, while the
capability of self-renew and differentiation of BMMSCs obtained from RA patients significantly
reduced compared with that from healthy people , which are also affected by age . It
indicates that the additional BMMSCs are demanded to remedy the flaws in RA. Fortunately,
BMMSCs lack the expression of major histocompatibility complex class II and immune
stimulating molecules, even under stimulation are still nonimmunogenic, so BMMSCs can escape
immune surveillance and won't cause graft rejection .
To date, in consideration of that RA is a systemic disease, the approaches that using
allogeneic BMMSCs grafting for systemic treatment on RA were mainly performed via intravenous
or intraperitoneal injection , while the localized administration is rarely attempted
. Once the BMMSCs are transplanted into articular cavity, the cells mainly obtain the
nutrition and oxygen from the synovial fluid , and the inflammatory microenvironment will
certainly hamper the cell viability. To address above problem, microfracture through the
nonweight-bearing area is created. In practice, the penetration from subchondral bone marrow
causes the formation of blood clot, which allows endogenous BMMSCs, various cytokines,
oxygen and nutrients to access the channel , and the implanted BMMSCs can enter bone
marrow to perform immunosuppressive function meanwhile.
Three-dimensional scaffold can provide a relatively stable space for the expansion of BMMSCs,
and thermosensitive hydrogels are one of the vastly applied bio-scaffold in tissue engineering,
including cartilage repair, bone regeneration and cardiac restoration . Among them, the
thermogel based on poly(D,L-lactide-co-glycolide)-block-poly(ethylene
glycol)-block-poly(D,L-lactideco-glycolide) triblock copolymers (PLGA-b-PEG-b-PLGA) has shown outstanding potential for
clinical applications due to their excellent tunable gelation behavior, biocompatibility and
biodegradability [27,28]. In addition, our previous work confirmed that the PLGA-b-PEG-b-PLGA
thermogel supports excellent adhesion and proliferation of BMMSCs . In this work, the influence
of intra-articular transplantation of exogenous BMMSCs entrapped in PLGA-b-PEG-b-PLGA
thermogel accompanying with endogenous MSCs from bone marrow after microfracture on
Fig 1. Schematic illustration of possible mechanisms for remission of collagen-induced arthritis.
systemic immunosuppression and protection of surrounding cartilage under arthritic condition
was clarified in rat model of collagen-induced arthritis (CIA), described as Fig. 1. It aimed to
explore a novel procedure to make up for the deficiency of current clinical treatments.
PLGA-b-PEG-b-PLGA triblock copolymer (number-average molecular weight (Mn, NMR) =
4200 g/mol, LA/GA = 75:25, mol/mol) was synthesized as our previous works . Percoll
density gradient, 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and
interleukin-1 (IL-1), tumor necrosis factor- (TNF-) and anti-collagen type II antibody
(antiCOL-II Ab) enzyme-linked immunosorbent assays (ELISAs) kit were purchased from
Sigma-Aldrich, USA. Chick type II collagen (COL-II) and complete Freund's adjuvant (CFA) were acquired
from Chondrex, USA. FITC-conjugated anti-CD3, PE-conjugated anti-CD4, and APC-conjugated
anti-CD8 mAbs were obtained from eBioscience, San Diego, CA. Dimethyl sulfoxide (DMSO) was
stored over calcium hydride (CaH2) and purified by vacuum distillation with CaH2.
Animal Models of CIA
As shown in Fig. 2, the protocol was approved by the Animal Care and Use Committee of
Second Hospital, Jilin University, and all efforts were made to minimize suffering. 36 male
Fig 2. Schematic chronogram of experimental design.
Sprague-Dawley (SD) rats (200 20 g, 6 weeks) were supplied by the Experimental Animal
Center of Jilin University. After adaptive feeding for a week, 36 SD rats were divided into 4
groups referred to as CON, BLA, GEL and BMC. Detailed information was described in
Table 1. All rats received a subcutaneous injection of the emulsion prepared by mixing
100.0 g of COL-II in 0.1 N acetic acid with 50.0 g of CFA at the base of tail. An additional
immunization boost was given at day 21 by half the dosage of emulsion at the base of tail.
SD rats of BLA, GEL and BMC groups (n = 27) underwent operations at one week post boost
injection. In brief, rat was anaesthetized with 3% (w/v) pentobarbital sodium (30.0 mg/kg) and
performed a longitudinal cut via a medial approach of left knee. The patella was dislocated and
the joint was flexed to exposure the tibial plateau. And then, a hole (diameter = 1 mm) was
drilled in the center of tibial plateau until the articular cavity was extended to subchondral
bone marrow. Finally, the patella was reset and the wound was closed in layers. The rats of
CON group underwent sham operation without drilling. Animals were allowed to move freely
after surgery. The rats received gentamicin by intramuscular injection (1.5 mg/kg) each day for
3 times post-operatively, and no rats were found with skin infection or dead during
Extraction and Culture of BMMSCs
Bone marrow was harvested from the tibia and femur of a male immature SD rat (50 g, 3
weeks). The mononuclear cells were isolated by percoll density gradient, and plated on culture
dish in low-glucose Dulbecco's modified Eagle's medium (LG-DMEM) supplemented with
10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin at 37C.
Non-adherent cells were removed 4 days later, and the attached BMMSCs were harvested. When cells
grew to * 80% saturation, the adherent cells were digested with 0.25% trypsin/EDTA at 37C
for 3 min and passaged, and the 3rd passage of BMMSCs were readied for use.
Administration of BMMSCs Loaded in PLGA-b-PEG-b-PLGA Thermogel
Cell therapy with allogeneic BMMSCs was performed 3 days after the surgery. Rats were
treated with the suspension of 5 106 allogeneic BMMSCs mixed in 100.0 l of
PLGA-b-PEG-bPLGA copolymer solution in phosphate-buffered saline (PBS) at 4C (20 wt.%; noted as BMC,
n = 9) or treated with 100.0 l of PLGA-b-PEG-b-PLGA thermogel alone (noted as GEL,
n = 9). The group treated with 100.0 l of PBS was named as blank (BLA, n = 9), and the group
without surgery treated with 100.0 l of PBS was named as control (CON, n = 9). The groups
were described in detail in Table 1. Rats were monitored for the signs of arthritis onset, and the
disease scores of CIA were assessed weekly using the previously described procedures on a
scale of 0 3 based on the level of erythema, swelling or joint rigidity, where 0 = no swelling,
1 = slight swelling and erythema, 2 = pronounced edema, and 3 = joint rigidity. Each limb of a
rat was scored separately, the total score of each rat was calculated and presented as the average
value by group (Arthritis Index) .
COL-II-Specific Proliferation Lymphocytes Derived from Draining Lymph
Node (DLN) and Spleen
The rats were sacrificed at 4, 8 or 12 weeks post-transplantation. Lymphocytes from DLN and
spleen were isolated at the time of sacrifice, and cells were supplemented and cultured as
previously described to measure specific cell proliferation . In brief, each culture was performed
for 2 105 lymphocytes/well in 96-well plate, and 200.0 l of complete LG-DMEM was added
in triplicate. CD11c+ dendritic cells were separated from spleen of the same rat, and were
pretreated with mitomycin C to prohibit their own proliferation, then added as antigen-presenting
cells with equal number of lymphocytes with the stimulation of COL-II at a concentration of
5.0 mol or otherwise. The lymphocytes cultured only with non-T cells served as negative
control. The plates were incubated in a humidified atmosphere of 5% CO2 at 37C for 72 h, and
then 20.0 l of MTT was added to each well to co-culture for 4 h. The supernatant was
removed, and 150.0 l of dimethyl sulfoxide was added. The absorbance of above medium was
measured at 490 nm on a Bio-Rad 680 microplate reader (Model 550, Hercules, CA, USA).
Flow Cytometry Analysis of T Lymphocyte Subsets
1.0 ml of peripheral blood was collected from the tail vein of SD rat with an Eppendorf tube
containing 50.0 l of heparin sodium solution (1000.0 IU/ml) at the time point of sacrifice (i.e.,
week 4, 8 and 12). Flow cytometry analysis was performed to detect T lymphocyte subsets in
peripheral blood using FITC-conjugated anti-CD3, PE-conjugated anti-CD4 and
APC-conjugated anti-CD8 mAbs. Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences,
USA), and the data were collected for 10,000 events per sample with CellQuest Pro software
(BD Biosciences, USA).
Measurement of Serum Cytokines and Specific Anti-Collagen Antibody
Peripheral blood was configured to collect the upper serum for the detection of cytokines,
IL1, TNF- and COL-II Ab by ELISAs following the instructions of ELISA assay kits. The
concentration of each protein was calculated from the standard curve.
Macroscopic and Histological Assessment
At 4, 8 and 12 weeks post-transplantation, the rats were sacrificed, and both the distal femurs
and surrounding synovium were isolated. The femurs were carefully examined and
photographed. After gross examination, distal femurs were collected and fixed in 4% (w/v)
paraformaldehyde, and decalcified and embedded for paraffin-sectioning (5 m). Hematoxylin and
eosin (H&E) staining was performed to evaluate the situation of surrounding synovial
membrane and cartilage. The morphological features of the synovium were assessed in H&E-stained
slices according to the criteria described in Table 2 . Three sections from each sample were
randomly chosen and scored by two blinded observers, and the possible maximum score is 9.0.
As depicted in Table 3, the modified ORASI score system was applied to evaluate the cartilage
status microscopically . In addition, the thickness of hyaline cartilage (HC, zone from
cartilage surface to tidemark) and calcified cartilage (CC, zone from tidemark to surface of
subchondral bone plate) were measured with imagine analysis software of Nano Measurer 1.2, and
the ratios of thickness of HC to CC were also calculated.
2. Slight enlargement (two to three cell layers). Giant cells are very rare
3. Moderate enlargement (four to five cell layers). Some giant cells or lymphocytes
4. Strong enlargement (more than six cell layers). Giant cells and lymphocytes are frequent
B. Inflammatory infiltration
2. Slight inflammatory infiltration (diffusely located single cells and small perivascular
3. Moderate inflammatory infiltration (perivascular and/or superficial lymphatic aggregates, and
4. Strong inflammatory infiltration (lymphatic follicles with germinal center and/or confluent subsynovial lymphatic infiltration)
2. Slight synovial stroma activation (low cellularity with slight edema, slight fibrosis with some fibroblast, no giant cells)
3. Moderate synovial stroma activation (moderate cellularity with a moderate density of
4. Strong synovial stroma activation (high cellularity with dense distribution of fibroblasts and
score = 2
score = 3
score = 2
score = 3
All experiments were performed at least three times, and the data were expressed as
means standard deviation (SD). The statistical analysis was performed with the software
SPSS 13.0 (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.
Prevention of Severe Tissue Damage in CIA Models by BMMSCs
In this work, 36 SD rats were immunized with COL-II to generate CIA models. One week after
boost immunization, 27 SD rats were randomly selected to perform the operation and divided
into 3 groups (BMC, GEL, BLA), the remaining 9 SD rats were noted as CON group. Then the
BMC group accepted the administration of 5 106 allogeneic BMMSCs mixed in 100.0 l of
PLGA-b-PEG-b-PLGA copolymer solution, and the GEL group accepted equal volume of
thermogel alone, the group treated with 100.0 l of PBS was named as BLA. The signs of CIA with
different degrees (Fig. 3A D) were observed as time goes on, and the mean disease score of all
CIA models was 6.6 at 12 weeks post cell therapy.
After the combination of microfracture and transplantation of thermogel-encapsulated
bone marrow mesenchymal stem cells, the BMC group showed significantly mildest symptoms
of RA with disease score at 3.9 0.4 compared with all the other groups (i.e., CON, BLA and
GEL). In addition, both the BLA (7.2 0.3) and GEL (7.1 0.4) groups exhibited relatively
slighter symptom of swollen joint in contrast with CON group (8.2 0.4) (Fig. 3E).
BMMSCs-Induced Hyporesponsiveness of Lymphocytes
The proliferation experiment of lymphocytes revealed that the treatment with allogeneic
BMMSCs induced the obvious hyporesponsiveness of lymphocytes from DLN (Fig. 4A) and
1. Slight surface irregularities
2. Moderate surface irregularities
3. Severe surface irregularities
4. Clefts/fissures into transitional zone (one-third depth)
5. Clefts/fissures into radial zone (two-thirds depth)
6. Clefts/fissures into calcified zone (full depth)
7. Fibrillation and/or erosion to transitional zone (one-third depth)
8. Fibrillation and/or erosion to radial zone (two-thirds depth)
9. Fibrillation and/or erosion to calcified zone (full depth)
1. Increase or slight decrease
2. Moderate decrease
3. Severe decrease
4. No cells present C. Chondrocyte cloning 0. Normal
1. Several doublets
2. Many doublets
3. Doublets and triplets
4. Multiple cell nests doi:10.1371/journal.pone.0120596.t003 Score score = 0
spleen (Fig. 4B) of immunized rats at 4, 8 or 12 weeks, whereas the lymphocytes from other
groups proliferated markedly when stimulated with COL-II under the same conditions
(p < 0.05). In addition, the rats in BLA and GEL groups also exhibited remarkable
immunosuppressive characters in DLN experiment (p < 0.05), but it was less obvious toward
Suppression of CD4+ T Lymphocytes Detected by Flow Cytometry
The subsets of T lymphocytes including CD3+, CD4+ (inducer/helper) and CD3+, CD8+
(cytotoxic/suppressor) in peripheral blood were measured by flow cytometry (Fig. 5). The
result revealed that the ratio of CD4+ to CD8+ lymphocytes, reflecting the balance of
immunoregulatory cells, in BMC group was lower than that in other groups (p < 0.05). In addition, the
BLA and GEL groups exhibited relatively less disparity compared with CON group. A upward
trend of the ratio of CD4+ to CD8+ in BMC group was observed as time goes on, indicating
fluctuant therapeutic effect after the combination treatment and a slow progression of CIA
after 4 weeks post-treatment.
Down-Regulated Expression of Inflammatory Cytokines Following
Treatment with BMMSCs
The mechanisms underlying the decrease in severity of CIA following operation and the
transplantation of BMMSCs were deliberated, and the effect on the production of inflammatory
Fig 3. Representative photos of different degrees of CIA severity (A D); disease scores in individual male SD rats after immunization with type II
collagen (COL-II) and treatment with allogeneic BMMSCs (E). Each limb of a rat was scored separately, and the average score for each animal
factors that are closely linked to synovium inflammation was also evaluated. The results
showed that the transplantation of BMMSCs significantly reduced the expression of
inflammatory cytokines, such as IL-1 and TNF- (Fig. 6A and 6B). As previously reported, the antibody
against collagen-rich synovium is involved in the pathogenesis of CIA . In this work, the
administration of BMMSCs resulted in the reduced serum levels of COL-II Ab (Fig. 6C),
suggesting that the local anti-inflammatory effect of administrated BMMSCs was accompanied
with the down-regulation of systemic inflammatory response.
Reduction of Synovial Hyperplasia and Cartilage Damage
Macroscopic appearance of joint. The macroscopic appearance of joint can reflect the
degree of cartilage destruction intuitively. In this study, the results demonstrated that there was
no cartilage lesion at 4 weeks in any groups. However, the gross findings in the control group
displayed the broad areas of cartilage damage with irregular surface fibrillation at 8 weeks,
which was also found in BLA and GEL groups at 12 weeks. The BMC group demonstrated the
best outcomes and showed generally intact surfaces, even if slight surface fibrillation was
revealed at 12 weeks (Fig. 7).
Histological analysis of synovium and cartilage. No inflammatory cells infiltrated the
fibrous and adipose tissues underlying the synovial lining of the synovial fringes, and minimal
hyperplasia of the synovial lining were observed in the BMC group. The histopathological
scores of synovium were 0.8 0.1, 1.6 0.1 and 1.8 0.3 at 4, 8 and 12 weeks, respectively. The
result indicated the absence of inflammatory reaction and slight synovial hyperplasia.
However, the hyperplasia of synovial lining and inflammatory cells suffusing in synovium in CON
group with histopathological scores of 4.8 0.8, 5.4 0.4 and 6.3 0.2 at 4, 8 and 12 weeks,
respectively, were observed throughout the detection stage. At the same time, the same
symptoms were observed at the later period of BLA with histopathological scores of 3.3 0.4 and
5.1 0.4 at 8 and 12 weeks, respectively, and GEL groups with those of 3.5 0.4 and 5.7 0.3
at 8 and 12 weeks, respectively (Fig. 8).
The cartilage histological results in CON group revealed the progression of CIA up to the
deep zones of the cartilage layers with high OARSI score. The results in both BLA and GEL
groups also demonstrated the slower progression of CIA changes with minor cartilage
Fig 4. Relative proliferation of lymphocytes isolated from DLN (A) and spleen (B) of CIA rats
stimulated by 5.0 mol of COL-II after treatment with BMMSCs. Data are represented as means SD
(n = 3).
destruction. The sample in BMC group exhibited no surface fibrillation and minimal degree of
cartilage lesion (Fig. 9A and 9B). The thinning of cartilage during RA progression could occur
from both directions by both surface degradation and the increase in cartilage calcification or
hypertrophy from the cartilage/bone interphase . The thickness of calcified cartilage in
these rats of CON, BLA and GEL groups with the tidemark moving closer to the articular
surface were greater than that in the rats of BMC group (Fig. 9C). In short, these results indicate
that the implantation of BMMSCs loaded in PLGA-b-PEG-b-PLGA thermogel exhibits
excellent anti-inflammatory and cartilage protective effect.
Fig 5. Flow cytometry analysis of CD3+ T lymphocyte subsets in peripheral blood analyzed using FITC-conjugated anti-CD3, PE-conjugated
antiCD4, and APC-conjugated anti-CD8 mAbs.
As above mentioned, RA is a systemic disease. If it is not treated properly, RA can eventually
lead to the irreversible severe articular cartilage damage . The patients who want to get rid of
unbearable pain need joint plasty or joint fusion, and one of the most important remedial
measures is to prevent the cartilage destruction . MSCs have attracted more and more attention
due to the excellent function of cartilage repair and immune-regulatory. Intra-articular
transplantation is the most immediate treatment approach for arthritis and other joint disorders.
However, the localized cell therapy can avoid some problems, such as unnecessary organs
involvement and tumorigenic issues , and can also reduce the total amount of cells used in
the treatment. Recently, it has been proven that the full-thickness defect extending into the
marrow cavity can recruit intrinsic BMMSCs to compensate for a deficiency in the number or
function of MSCs that occur in the injured tissues . In addition, the stimulation of marrow
is based on the provocation of inflammation, which leads to the mobilization of endogenous
bone marrow-derived MSCs . In this work, a new way to prevent progressive RA from
severe cartilage damage was attempted. Firstly, a channel linking bone marrow to articular cavity
was created to enhance the circulative capacity of intrinsic BMMSCs, and then the exogenous
BMMSCs encapsulated in PLGA-b-PEG-b-PLGA thermogel were implanted. Under the
stimulation of arthritic condition, the mobilized BMMSCs can secrete various paracrine factors to
Fig 6. Concentrations of IL-1, TNF- and anti-COL-II Ab in serum analyzed by ELISA. Data are
represented as means SD (n = 3).
regulate the immune response locally or through hematologic system and node pathways to
exert their immune regulatory function systemically.
Fig 7. Typical appearance of articular cartilage at different time points after implantation.
Joint swelling is a common symptom for RA patients, which is mainly caused by the
synovial hyperplasia and joint effusion . In this study, the count indexes of swollen joint revealed
that the vast majority of immunized rats developed irreversible bone or cartilage erosions
except for BMC group, whose degree of disease score was lower than those of non-treatment
groups, while higher than normal rats. It indicated that the treatment of BMMSCs could
inhibit, but could not completely eliminate synovial hyperplasia, which had been confirmed by the
histopathological analysis that revealed the reversible symptom of synovial inflammation. The
operation, which leads to the convenient situation for the recruitment of endogenous
BMMSCs, can ameliorate the condition of CIA in some extent.
In order to investigate the pathway of local BMMSCs therapy, the specific stimulation of
lymphocytes and splenocytes by COL-II was assessed. The outcomes proved that BMMSCs
behaved their immunosuppressive function through lymph metastasis or blood circulation with
an obvious declining inhibitory effect. The flow cytometry analysis ingenuity demonstrated
that the transplantation of BMMSCs can influence the systemic situation of inflammatory
Fig 8. Longitudinal section of distal femur indicating the region of interest (black box and yellow box for synovium and cartilage, respectively) (A);
synovium was stained with H&E (scar bar = 100 m) (B), and the histopathological score was evaluated at 4, 8 and 12 weeks post-transplantation
response, and the ratio of CD4+ to CD8+ T lymphocytes significantly decreased during the
experiment performance in BMC group compared with that of other groups. Whereas, the ratios
of CD4+ to CD8+ T lymphocytes in BLA and GEL groups were lower than that in CON group,
which may account for the intervention of intrinsic BMMSCs.
The proinflammatory cytokines, such as IL-1, may affect the chondrogenic and anabolic
ability of cartilage, and are considered to display catabolic effects and stimulate proteinases.
Subsequently, they may result in extracellular cartilage matrix degradation that impedes
cartilage repair, maturation and lateral integration . TNF- stimulates the proliferation of
synovial cells, and induces the release of other pro-inflammatory cytokines, leading to joint
destruction . Anti-COL-II Ab, which is secreted by activated B lymphocytes under the
stimulation of COL-II, is a key index indicating the severity of collagen induced arthritis .
The decreased levels of IL-1, TNF- and anti-COL-II Ab in serum suggested that the
transplantation of BMMSCs significantly attenuated the inflammatory situation through the
downregulation of pro-inflammatory cytokines and antigen specific antibody.
Fig 9. H&E staining of cartilage sections (scar bar = 100 m) (A), the modified OARSI score of microscopic observation (B) and the ratio of the
thickness of hyaline cartilage to calcified cartilage were evaluated at 4, 8 and 12 weeks post-transplantation (C).
As we all know, synovial fibroblasts (SFs) are one of the most abundant resident cells in
synovium. In the synovium of RA patients, the abnormal proliferation of SFs occurs by either
acquired stimulation or the recruitment of variable ancestral cells, and amount of evidences
support that expansion of SFs contributed to the pathogenesis of RA . SFs can respond to a
variety of cytokines, particularly TNF-, and SFs can secrete various inflammatory mediators
that may result in cartilage destruction [44,45]. The cytokine-induced cascade responses lead
to the up-regulation of the synthesis of cytokines, pro-angiogenic factors, chemokines and
factors associated to enhanced invasiveness and cartilage destruction . Therefore, there is a
closely connection between cytokines, synovium and cartilage. A major objective of this paper
is to check the efficacy of MSCs therapy on cartilage protection, and the basic performance of
cartilage destruction can be examined by the direct observation of joint surface and
histopathological staining. It turned out that the synovial hyperplasia was observed at 4 weeks in CON
group, and 8 weeks in BLA and GEL groups, simultaneously, the cartilage destruction was
observed at 8 weeks in CON group, and 12 weeks in BLA and GEL groups. Fortunately, there was
no synovial hyperplasia or cartilage destruction in BMC group over the entire detection period.
All the results were future confirmed by the H&E staining of surrounding synovium and
cartilage. Apart from the synovial hyperplasia, the infiltration of inflammatory cells was found in
the deep layer of synovium of CON, BLA or GEL group except for BMC group. Those results
indicated that the intrinsic BMMSCs can delay the progress of synovial hyperplasia and
cartilage destruction, while the transplantation of BMMSCs in thermogel can inhibit synovial
hyperplasia and the infiltration of inflammatory cells and thereby ameliorate the cartilage
There may be two possible mechanisms to explain the above declaration. Firstly, a previous
study reported that MSC-differentiated chondrocytes owned the same immunological
characters as the undifferentiated MSCs. It suggests that MSC-chondrocytes might suppress
inflammatory factors and prevent re-destruction while exerting their repairing effects once being
implanted into RA joints . Another study revealed that the differentiated MSCs, such as
fibroblasts, could inhibit T cell proliferation in vitro, while showed no immunosuppressive effect
in vivo . Actually, MSCs responding to the irritation of inflammation can be influenced by
the presence of inflammatory factors. The addition of TNF- can reverse the
immunosuppressive effect of MSCs on T cells proliferation. It suggests that the interactions between MSCs and
arthritic microenvironment are reciprocal . Beyond that, the channel between articular
cavity and subchondral bone marrow is blocked by regenerative tissue, which can certainly
impede the interaction between articular cavity and bone marrow. It may explain why the
immunosuppressive effect of BMMSCs cannot persist for a long period. In previous studies, the
intrinsic MSCs were rarely investigated rather than being utilized. Despite the less remarkable
effects than those of exogenous BMMSCs admission, to a certain extent, the recruitment of
endogenic BMMSCs alleviate the RA-linked symptoms, down-regulate the hyporesponsiveness
of lymphocytes and slow the progress of RA, which all are verified by the histological
examination. Nevertheless, the mechanisms remain further investigation.
Further research on the BMMSCs-located microenvironment and regulatory mechanism
will be of central importance. The advanced studies should focus on how to take advantage of
BMMSCs accompanied with the endogenous progenitor cells to modulate the local
microenvironment for the purposes of protection or repair of articular cartilage. In addition, the MSCs
from different sources, such as umbilical cord and adipose, also exhibit excellent therapeutic
benefits on RA , and should be employed to optimize the above treatment program.
In this study, a novel strategy by the combination of microfracture and local BMMSCs
transplantation with a biodegradable PLGA-b-PEG-b-PLGA thermogel for the treatment of CIA
were presented. Notably, the endogenous from microfracture and implanted exogenous
BMMSCs can synergistically reduce the ratio of CD4+ to CD8+ T lymphocytes in peripheral
blood and result in decreased levels of inflammatory cytokines. Moreover, the inhibitory effects
on the proliferation of antigen specific lymphocytes and local articular inflammation, as well as
the protective role on cartilage of the BMMSCs were revealed. While, the recruitment of
endogenic BMMSCs through microfracture exerted lower effectiveness in these regards. Based on
the above results, the MSCs transplantation combined with microfracture will present great
hope in relieving the disease burden of RA patients.
Conceived and designed the experiments: FC XC JW YW. Performed the experiments: HL JD
MY YZ. Analyzed the data: HL JD. Contributed reagents/materials/analysis tools: FC XC JW
YW. Wrote the paper: HL JD.
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