Lamo‑Espinosa et al. J Transl Med (2016) 14:246
Intra‑articular injection oftwo
dierent doses ofautologous bone marrow
mesenchymal stem cells versushyaluronic
acid inthe treatment ofknee osteoarthritis:
multicenter randomized controlled clinical
trial (phase I/II)
José M. Lamo‑Espinosa1†, Gonzalo Mora1†, Juan F. Blanco2,3, Froilán Granero‑Moltó1,3,4,5,
Jorge M. Nuñez‑Córdoba5,6,7, Carmen Sánchez‑Echenique1, José M. Bondía8, Jesús Dámaso Aquerreta8,
Enrique J. Andreu3,4, Enrique Ornilla9, Eva M. Villarón3,10,12, Andrés Valentí‑Azcárate1, Fermín Sánchez‑Guijo3,10,12,
María Consuelo del Cañizo3,10,12, Juan Ramón Valentí‑Nin1 and Felipe Prósper3,4,5,11*
Background: Mesenchymal stromal cells are a promising option to treat knee osteoarthritis. Their safety and useful‑
ness must be conﬁrmed and the optimal dose established. We tested increasing doses of bone marrow mesenchymal
stromal cells (BM‑MSCs) in combination with hyaluronic acid in a randomized clinical trial.
Materials: A phase I/II multicenter randomized clinical trial with active control was conducted. Thirty patients
diagnosed with knee OA were randomly assigned to intraarticularly administered hyaluronic acid alone (control), or
together with 10 × 106 or 100 × 106 cultured autologous BM‑MSCs, and followed up for 12 months. Pain and func‑
tion were assessed using VAS and WOMAC and by measuring the knee motion range. X‑ray and magnetic resonance
imaging analyses were performed to analyze joint damage.
Results: No adverse eﬀects were reported after BM‑MSC administration or during follow‑up. BM‑MSC‑administered
patients improved according to VAS during all follow‑up evaluations and median value (IQR) for control, low‑dose and
high‑dose groups change from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 4 (3, 5), 2 (1, 3) and 2 (0,4) respectively at 12 months (low‑
dose vs control group p = 0.005 and high‑dose vs control group p < 0.009). BM‑MSC‑administered patients were also
superior according to WOMAC, although improvement in control and low‑dose patients could not be signiﬁcantly
sustained beyond 6 months. On the other hand, the BM‑MSC high‑dose group exhibited an improvement of 16.5 (12,
19) points at 12 months (p < 0.01). Consistent with WOMAC and VAS values, motion ranges remained unaltered in the
control group but improved at 12 months with BM‑MSCs. X‑ray revealed a reduction of the knee joint space width in
the control group that was not seen in BM‑MSCs high‑dose group. MRI (WORMS protocol) showed that joint damage
decreased only in the BM‑MSC high‑dose group, albeit slightly.
© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
†José M. Lamo‑Espinosa and Gonzalo Mora contributed equally to this
11 Department of Hematology, Clínica Universidad de Navarra, Avenida
Pío XII 36, 31009 Pamplona, Navarra, Spain
Full list of author information is available at the end of the article
Page 2 of 9
Lamo‑Espinosa et al. J Transl Med (2016) 14:246
Osteoarthritis (OA) is a chronic disease involving pro-
gressive degeneration of the articular cartilage and sub-
chondral bone, accompanied by synovitis . Due to its
avascular nature and the limited self-renewal capacity
of chondrocytes, adult articular cartilage presents lim-
ited repair capability . Current treatment options for
articular cartilage injury and osteoarthritis are aimed to
relieve inﬂammation and pain, but have no eﬀect on the
natural progression of the disease . To date, in severe
cases of knee OA, knee replacement is the only therapeu-
tic option .
During the last two decades focal cartilage defects
have been treated using cell therapy and tissue engineer-
ing approaches. In this context, autologous chondro-
cyte implantation (ACI) or matrix-induced autologous
chondrocyte (MACI) implantation techniques have
been applied with promising results, although the large
non-contained cartilage defects found in OA and its
own pathogenesis cannot be treated using ACI or MACI
[5–7]. e use of intraarticular injections of mesenchy-
mal stromal cells (MSCs) may represent some advantages
over chondrocytes in patients with OA. First, because
of their ability for self-renewal, the number of cells that
can be obtained is increased without cartilage donor site
morbidity and with reduced cost [6–8]. Second, MSCs
are responsible for the normal turnover and mainte-
nance of adult mesenchymal tissues, including cartilage,
and has been suggested that the number of MSCs pre-
sent in the subchondral bone decreases with age and OA
grade, suggesting that such MSCs deﬁcit could prime the
degenerative process [9–12]. It has also been proposed
that during tissue injury MSCs migrate to participate in
the reparative process, giving MSCs a potential therapeu-
tic value when added exogenously [13, 14]. Additionally,
cultured MSCs induce in vitro chondrocyte prolifera-
tion and extracellular matrix protein synthesis, including
aggrecan and type II collagen, which support their criti-
cal role in cartilage tissue repair [15, 16].
ere is an increasing number of reports on the treat-
ment of OA using MSC, but these are methodologically
heterogeneous in dose, cell source, coadjuvants and cell
processing methods, which makes it diﬃcult to com-
pare the diﬀerent studies . In many cases, treatments
consist of the administration of bone marrow concen-
trates as a source of MSCs. However, it is well known
that only 0.001% of the mononuclear cells found in the
bone marrow could be considered as MSCs as deﬁned
by the ICRS in 2006. erefore, their number in a bone
marrow concentrate is very limited compared to that
obtained upon culturing MSCs [18–20]. Only a few stud-
ies using MSCs produced by good manufacture practices
(GMP) such as advanced cell-therapy products have
been reported [21–24]. In addition, there is a need to
explore the eﬀect of diﬀerent cell doses in a randomized
way to gain insight into the ideal conditions for knee OA
patients to take advantage of MSC therapy. For these rea-
sons, the purpose of this study was to randomly assess
the safety, feasibility and eﬃcacy of the intra-articular
injection of two diﬀerent doses of GMP-produced autol-
ogous bone marrow MSCs (BM-MSCs) with hyaluronic
acid (HA) in patients with knee OA.
Participants andstudy design
is is a phase I/II randomized clinical trial with active
control conducted between August 2012 and Octo-
ber 2014, involving the Clínica Universidad de Navarra
(Pamplona, Spain) and IBSAL-Hospital Universitario de
Salamanca (Salamanca, Spain). All the procedures were
approved by the Institutional Review Board of Nav-
arra and the Spanish Agency of Medicines and Medical
Devices (Nº EudraCT: 2009-017624-72, Clinical Trials.
gov identiﬁer: NCT02123368). All participants provided
written informed consent.
Criteria foreligibility ofpatients
Inclusion criteria were as follows: males and females aged
50–80, diagnosis of knee OA according to American
College of Rheumatology criteria, visual analogue scale
(VAS) joint pain ≥2.5, Kellgren–Lawrence radiological
classiﬁcation scale ≥2, body mass index between 20 and
35kg/m , and availability to be followed during the
study period; exclusion criteria were: previous diagnosis
of polyarticular disease, severe mechanical extra-articu-
lar deformation (>15° varus/15° valgus), systemic auto-
immune rheumatic disease, arthroscopy or intraarticular
inﬁltration in the last 6months, chronic treatment with
Conclusions: The single intraarticular injection of in vitro expanded autologous BM‑MSCs together with HA is a safe
and feasible procedure that results in a clinical and functional improvement of knee OA, especially when 100 × 106
cells are administered. These results pave the way for a future phase III clinical trial.
Clinical Trials.gov identiﬁer NCT02123368. Nº EudraCT: 2009‑017624‑72
Keywords: Bone marrow‑mesenchymal stromal cells, Knee osteoarthritis, Non‑surgical management,
Stem cell therapy
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Lamo‑Espinosa et al. J Transl Med (2016) 14:246
immunosuppressive or anticoagulant drugs, corticoster-
oids treatment in the 3 last months, nonsteroidal anti-
inﬂammatory drugs therapy in the last 15days, bilateral
knee OA requiring treatment in both knees, poorly con-
trolled diabetes mellitus, blood dyscrasias, and allergy to
HA or bird proteins.
Participants were assigned to comparison groups by
an unblinded computer-generated list, based on unre-
stricted randomization, which was maintained centrally
by staﬀ with no clinical involvement in the trial so no
center knew the treatment allocation of any patient until
the patient had been recruited into the trial.
ree groups were created:
Control group, constituted by patients who received a
single intra-articular injection of 60mg HA (Hyalone®)
in a ﬁnal volume of 4ml.
Low-dose BM-MSCs group, constituted by patients
who received a single intra-articular injection of 10×106
autologous cultured BM-MSC in 1.5ml Ringer’s lactate
solution, followed by an intraarticular injection of 4ml
High-dose BM-MSCs group, constituted by patients
who received a single intra-articular injection of
100×106 autologous cultured BM-MSCs in 3ml Ring-
er’s lactate solution, followed by an intraarticular injec-
tion of 4ml HA.
Sample size calculation
We estimated that a sample size of ten patients per group
was required to detect an eﬀect size of 0.6 with a power
of 80 %, assuming a balanced allocation to treatment
groups, and a 5% type I error probability.
BM-MSCs were generated under good manufacturing
practice conditions (GMP) with standard operating pro-
cedures. Brieﬂy, bone marrow (100 ml) was harvested
from the pelvic bone (iliac crest) under sterile condi-
tions. e mononuclear cell fraction was isolated by
Ficoll density gradient centrifugation (Ficoll-Paque, GE
Healthcare Bio-Sciences AB, Uppsala, Sweden). Cells,
ranging between 20 × 106 and 60 × 106, were subse-
quently seeded in 175cm2 ﬂasks with growth medium,
which consisted of αMEM without ribonucleosides
(Gibco, Life Technologies, Carlsbad, CA, USA) supple-
mented with 5% platelet lisate, 2units/ml heparin, peni-
cillin–streptomycin at 1% (Gibco) and 1ng/ml human
ﬁbroblast growth factor (bFGF) (Sigma-Aldrich, St.
Louis, MO, USA). e ﬂasks were maintained in culture
at 37°C in 5 % CO2 atmosphere. e growth medium
was changed every 3–4 days. About 10–15 days later,
colonies were formed and the cells were split with Try-
pLE Select™ (Life Technologies) and seeded at 3000–
5000 cells/cm2. Once 70–80% conﬂuence was reached,
cells were split again and cultured until they were
available at the amounts required to be administered
to patients. Finally, cells were harvested with TrypLE
Select™, washed three times with PBS and resuspended
in Ringer’s lactate buﬀer (Grifols, Barcelona, Spain) con-
taining 1% human albumin (Grifols), to be administered
within 24h of harvesting of the cells. Cells were charac-
terized according to ISCT criteria. Cells were then ana-
lyzed by ﬂow cytometry (FACSCalibur, BD Biosciences,
San José, CA, USA) with the appropriate antibodies (BD
Biosciences) to conﬁrm expression of surface markers
CD90, CD73 and CD44, as well as absence of CD34 and
Cell injection was performed without radiographic guid-
ance through a lateral patellar approach by three diﬀer-
ent orthopaedic surgeons from both involved centers
(Additional ﬁle1: Figure S1), 3–4weeks after the iliac
crest biopsy had been performed. In 90% of the patients,
cells were administered within the ﬁrst hour after being
harvested. For this purpose, a 19G needle was used in
two consecutive intraarticular injections. In the ﬁrst one,
10×106 (low dose) or 100×106 (high dose) BM-MSCs
were administered in 1.5 and 3 ml doses respectively.
Subsequently, 4ml HA (Hyalone®) were injected using
the same via.
e occurrence of complications and/or adverse eﬀects
during the study was registered. In addition, the response
to the intra-articular infusion of HA with or without BM-
MSCs was assessed using the following procedures:
A goniometer-based evaluation of the articular range of
motion at baseline i.e. before treatment administration,
and 3, 6 and 12months after treatment.
Two scale-based methods Visual Analog Scale (VAS)
 and the Likert version of the Western Ontario and
McMaster Universities Osteoarthritis Index (WOMAC)
[26, 27], evaluated at baseline and 3, 6 and 12months
after treatment, to clinically assess pain and function.
VAS ranges from 0 (maximum relief, i.e., no pain) to 10
(no relief, i.e., maximal pain). WOMAC comprises three
subscores: pain, which includes 5 items; stiﬀness, with 2
items; and physical function, with 17 items. According to
previous literature, patients were considered WOMAC
responders when they reported an improvement of 20%
in at least two items together with an improvement of ten
points in the overall scale .
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Lamo‑Espinosa et al. J Transl Med (2016) 14:246
Rosenberg X-ray projections at baseline and 6 and
12months afterwards to provide a radiographic assess-
ment of the joint space width. A custom methacrylate
patient positioner was used to achieve a comparative
view (Additional ﬁle2: Figure S2).
A magnetic resonance imaging (MRI) study at baseline
and 6 and 12months after treatment. Two experienced
radiologists evaluated MRI images in a blinded manner
by assessing the number and location of the lesions, car-
tilage thickness, signal intensity, and subchondral bone
alteration and volume, following the Whole-Organ Mag-
netic Resonance Imaging Score (WORMS) protocol, in
which higher score values indicate more damage .
3T Magnetom TRIO equipment (Siemens, Erlangen,
Germany) was used following a protocol which included
an axial T1 weighted image (WI) with slice thickness of
5mm, coronal T1 WI (4mm), sagittal T1 WI (4mm),
sagittal T2 FS WI (4mm) and sagittal gradient echo 3D
e analyses were performed according to treatment
assignment, and all available data from all patients were
included in the analyses, following the intention-to-treat
principle. Descriptive data summaries are presented
as median [interquartile range (IQR)] or percentages.
Within each group, the comparison of each clinical
and radiographic endpoint between the value obtained
at 6 or 12months and the baseline value, i.e. the one
obtained immediately before the administration of the
treatment, was performed using the Mann–Whitney
U test. Changes in the same end points over time were
determined calculating the diﬀerences between the
measurements collected at the 6 or 12-month follow-up
visit and the baseline visit. Subsequently, comparisons
between treatment groups were carried out using the
Kruskal–Wallis test and the Mann–Whitney U test. All
tests were two-tailed. A p value of 0.05 was considered
to indicate statistical signiﬁcance, without adjustment
for multiple testing. All analyses were performed using
Stata 14 (StataCorp. 2015. Stata Statistical Software:
Release 14. College Station, TX: StataCorp LP) and IBM
SPSS Statistics 20 (IBM Corp. Released 2011. IBM SPSS
Statistics for Windows, Version 20.0. Armonk, NY: IBM
irty-two patients were assessed for eligibility, and were
consecutively randomized to treatment groups (Fig. 1).
Two patients who had been randomly assigned to the
control group withdrew consent and were excluded from
the trial. All the groups showed similar baseline charac-
teristics of age and body mass index. Patients in the three
groups showed an uneven distribution according to the
Kellgren–Lawrence scale but without statistical signiﬁ-
cance (p=0.585, Table1).
No serious adverse events or complications derived from
the procedures or treatments were noted. ere were
no clinically important trends in the results of physical
examination, vital signs and laboratory tests during the
study. Articular pain requiring anti-inﬂammatory treat-
ment during the ﬁrst 24h after inﬁltration was observed
in 1, 3 and 6 patients in the control, low-dose BM-MSC
and high-dose BM-MSC groups respectively. All patients
recovered completely without sequelae and no treatment
group-dependent diﬀerences were detected in the dose
of required anti-inﬂammatory drug or in the time that
passed until recovery.
Clinical assessment ofpain andfunction
VAS and WOMAC clinical scores were used in order to
obtain the best picture of how patients perceived their
own evolution. Evaluations were performed before the
administration of treatment and 3, 6 and 12 months
afterwards, and the results are summarized in Fig. 2,
Additional ﬁle3: TableS1 (VAS) and Table2 (WOMAC).
e patients that were solely given HA did not show
changes during follow up in their pain status according
to VAS (Fig.2; Additional ﬁle3: TableS1). Furthermore,
although they initially perceived some improvement
according to the WOMAC pain and physical function
subscores, this perception was not signiﬁcantly sustained
in the long term (Table2). Inatraarticular delivery of BM-
MSCs, specially when used at high dose, enabled patients
to perceive an improvement in their perception of pain in
their daily activity. On one hand, the VAS score value was
signiﬁcantly reduced upon treatment with low and high
BM-MSC doses at all follow-up times (Fig.2; Additional
ﬁle3: TableS1). Furthermore, treatment with 100×106
cells was associated with a signiﬁcant improvement in all
WOMAC subscores at 12months (Table2). It is impor-
tant to note that, when the overall WOMAC value at
12months was subtracted from the baseline value in each
patient, the median decrease in the score, i.e. the relief of
the symptoms, was notably larger if patients had been
treated with BM-MSCs [−6.5 (−19, 4), −14 (−27, 4), and
−14 (−15, −8), median (IQR), for control, low-dose and
high-dose BM-MSCs groups respectively]. us, only the
patients who had been treated with BM-MSCs met cri-
teria to be considered WOMAC responders in the long
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Lamo‑Espinosa et al. J Transl Med (2016) 14:246
Eect oftreatments onthe range ofknee motion
e knee ﬂexion and extension ranges of motion were
signiﬁcantly improved in the patients who were treated
with BM-MSCs and the eﬀect was seen earlier in patients
receiving the higher doses of BM-MSC. No improvement
was seen in patients receiving HA alone (Fig.3; Addi-
tional ﬁle4: TableS2).
Fig. 1 Study ﬂow diagram. Patients were screened in the two participating centers by using the inclusion and exclusion criteria
Table 1 Baseline characteristics ofpatients
Unless specied, data are presented as median [interquartile range (IQR)]. OA
osteoarthritis, K–L Kellgren and Lawrence grading scale of severity of knee OA
N 10 10 10
Age (years) 60.3 (55.1, 61.1) 65.9 (59.5, 70.6) 57.8 (55.0, 60.8)
Males, n (%) 7 (70) 4 (40) 8 (80)
BMI (kg/m2) 29.6 (26.2, 30.8) 27.1 (24.4, 31.2) 28.5 (25.8, 31.0)
Time since OA
diagnosis (years) 6 (2, 8) 9 (4, 12) 10 (7, 15)
K‑L 2, n (%) 4 (40) 1 (10) 3 (30)
K‑L 3, n (%) 2 (20) 2 (20) 3 (30)
K‑L 4, n (%) 4 (40) 7 (70) 4 (40) Fig. 2 VAS scores along the study. The median values of VAS in
the three groups before administration of treatments and 3, 6 and
12 months afterwards are presented. *p < 0.05; **p < 0.01 with
respect to the baseline value of the same group
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Lamo‑Espinosa et al. J Transl Med (2016) 14:246
Radiological andMRI ndings
e analysis of the knee joint space by X-rays during fol-
low up showed a borderline reduction in the control group
(p=0.05 at 12months), which was not observed in patients
treated with high dose BM-MSC (Table3; Additional ﬁle5:
TableS3). e assessment in the low dose group was not
possible because the baseline value was 0. ese results
suggest that BM-MSC may halt the progressive loss of car-
tilage observed in patients with OA despite the use of HA.
Consistent with the X-Ray results, the analysis of the MRI
following the WORMS protocol showed a reduction in the
score value during follow up (Table4). Patients treated with
high dose BM-MSCs showed a median improvement of 4
points at 12months, with an improvement of 22 points in
25% of patients, while there were no signs of improvement
either in the control or in the low BM-MSC group.
e interest in the clinical use of MSCs for the treatment
of knee OA has recently grown. However, the optimal
dose and source of cells, as well as the use of coadjuvants,
are not yet established. In the present clinical trial we used
two single doses of BM-MSCs, 10 and 100 × 106 cells,
coadministered with HA, and compared their eﬀects with
the single administration of HA in patients with knee OA.
Table 2 WOMAC score beforeadministration oftreatments
and3, 6 and12months afterwards
The values of each one of the three WOMAC subscales as well as the overall
WOMAC score at baseline and 3, 6 and 12months afterwards are presented.
Data are the median (IQR) of each group. Function means physical function.
*p<0.05, **p<0.01 with respect to the baseline value of the same group
WOMAC Time Control BM-MSCs
Pain Baseline 5.5 (5, 6) 7.5 (5, 9) 4.5 (4, 5)
3 months 3 (1, 3)* 3.5 (3, 7) 3 (2, 5)
6 months 2.5 (1, 5)* 3.5 (3, 7) 3.5 (2, 5)
12 months 2 (1, 6) 3.5 (3, 5) 2.5 (2, 4)*
Stiﬀness Baseline 2 (1, 3) 4 (2, 5) 2.5 (2, 4)
3 months 2 (1, 2) 2 (0, 4) 2 (1, 2)
6 months 0.5 (0, 2) 1.5 (1, 3)* 2 (1, 3)
12 months 2 (1, 2) 2 (1, 2)* 2 (1, 2)*
Function Baseline 21 (13, 24) 26.5 (23, 32) 19 (12, 25)
3 months 9 (7, 11)* 17.5 (8, 26) 10 (7, 18)
6 months 7.5 (2, 13)* 18 (10, 23) 14.5 (8, 17)
12 months 9.5 (5, 23) 17 (10, 20) 11 (9, 14)*
Overall Baseline 29 (19, 38) 37 (32, 42) 28 (16, 34)
3 months 12 (11, 14)* 25.5 (11, 37) 13 (11, 26)*
6 months 10 (4, 20)* 24 (13, 31) 20 (13, 23)
12 months 13.5 (8, 33) 21.5 (15, 26) 16.5 (12, 19)**
Fig. 3 Knee range of motion along the study. The median values
expressed in degrees of the goniometric measurements of the
knee ﬂexion (top) and extension (bottom) ranges of motion before
administration of treatments and 3, 6 and 12 months afterwards are
presented. *p < 0.05; **p < 0.01 with respect to the baseline value of
the high‑dose group. #p < 0.05 with respect to the baseline valued of
the low‑dose group
Table 3 X-ray measurement ofthe evolution of the knee
articular interline at6 and12months afterthe administra-
For each group of treatment, variation for knee joint space width, which was
measured in mm, was calculated by subtracting, for each patient of the group,
the value at 6 or 12months from the baseline value. Data are presented as the
median (IQR) of each group
Time Control BM-MSCs
6 months −3 (−6, 0) 0 (−1, 0) 0 (−1, 1)
12 months −4 (−18, 0) 0 (0, 3) 0 (−1, 2)
Table 4 WORMS score beforeadministration oftreatments
and6 and12months afterwards
The overall WORMS scores at baseline and 6 and 12months afterwards are
presented as the median (IQR) of each group. The evolution within each
treatment group at 12months is also presented, and was calculated by
subtracting for each patient the values at 12months from the corresponding
baseline values. Data are the median (IQR) of each group
Time Control BM-MSCs
Baseline 79 (41, 94) 75 (64, 107) 60 (53, 84)
6 months 78 (34, 107) 70 (57, 126) 53 (51, 90)
12 months 83 (25, 95) 90 (67, 140) 53 (46, 82)
12 months evolution −0.5 (−16, 15) 2.5 (−3, 25) −4 (−22, 2)
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Lamo‑Espinosa et al. J Transl Med (2016) 14:246
We found that the use of BM-MSCs resulted in a signiﬁ-
cant relief of pain symptoms in the long term. According
to the VAS scores, when BM-MSCs had been adminis-
tered, an improvement was seen from the earliest evalua-
tion and was maintained until the last one, at 12months,
at which time point the highest eﬀect was observed.
Interestingly, this pain reduction was independent of the
dose of BM-MSCs administered. On the other hand, no
signiﬁcant changes in VAS were detected in the control
group, and the value at 12months was similar to the one
registered before the administration of the treatments.
Accordingly, the analysis of the information provided by
WOMAC score conﬁrmed that BM-MSCs induced relief
of pain symptoms. It is interesting to note that, although
treatment with HA alone was able to reduce the WOMAC
score during the ﬁrst 6months, this improvement was not
sustained in the long term, but when patients received
BM-MSCs, a signiﬁcant reduction in WOMAC score
was detected at 12months. In addition, unlike what was
observed with VAS, only the high dose of BM-MSCs
showed an eﬃcient reduction in the WOMAC score.
Furthermore, it is notable that only patients treated with
high-dose BM-MSCs met the criteria to be considered
WOMAC responders .
e eﬀect of MSCs on pain improvement in knee OA
is controversial and the literature provides diﬀering
accounts. One metaanalysis and a comprehensive review
have been recently published on this topic. Xia etal. 
performed a metaanalysis by managing the results of
seven clinical trials, concluding that cell treatments were
not able to reduce pain scores. Unfortunately, the heter-
ogeneity in the methodology used in the diﬀerent stud-
ies, with diﬀerent cell production methods and dosage,
precludes these authors from drawing solid conclusions.
On the other hand, Rodríguez-Merchán  reviewed
25 articles that reported the use of intra-articular injec-
tion of MSCs in knee OA, ﬁnding that MSCs induce pain
relief and functional improvement in three randomized
clinical trials which, however, were not comparable to
ours methodologically. One of them used bone marrow
concentrate, another used peripheral blood progenitor
cells, while the third one used cultured autologous BM-
MSCs together with a high tibial osteotomy, which is a
surgical treatment with a well-known impact on pain
relief [31–33]. e number of clinical randomized tri-
als comparing diﬀerent treatment and dosage is limited.
In an interesting study, Orozco etal. [24, 34] reported
an improvement in pain and function with the use of
a single intra-articular injection of 40 × 106 cultured
autologous MSCs in twelve patients. In a more compa-
rable randomized clinical trial, using allogenic MSCs,
Vega etal. reported good clinical outcomes in pain con-
trol and function when comparing the use of a single
intra-articular injection of 40 × 106 cultured allogenic
MSCs against a single intraarticular injection of HA .
Osteoarthritis is not considered a classical inﬂammatory
arthropathy due to the absence of neutrophils in the synovial
ﬂuid and the lack of systemic manifestations of inﬂammation
. However, it is frequently associated with inﬂammation
signs and symptoms such as joint pain, swelling and stiﬀ-
ness, leading to signiﬁcant functional impairment and dis-
ability . e improvement in pain scores together with
the mild eﬀect on function and MRI scores suggests that the
positive eﬀect of BM-MSCs that we have observed may rely
on their paracrine function. In support of this notion, MSC
antiinﬂammatory properties have been correlated with pain
reduction elsewhere [37–40]. In addition, the reduction
in pain scores may explain the positive changes in ﬂexion
and extension. Although such changes are small, it must be
noted that a limitation of only a few degrees in ﬂexo-exten-
sion may severely compromise the daily functional activity.
ese improvements together with the ﬁndings in the image
analyses, suggest that MSC-based therapies may be indicated
in asymptomatic patients with mild OA grade, in whom the
injected MSCs could be more eﬀective through their parac-
rine function when a healthier cartilage is still present.
e maintenance of the knee joint space width has been
related to an appropriate cartilage thickness . Unlike
what happened in the patients that were treated with HA
only, who experienced a reduction of this space over the
time of the study, the space width was preserved when
BM-MSC were also administered, even though the results
obtained in the patients that had received the low dose
must be taken cautiously since the baseline value in 25%
of them was already zero, which precludes suitable follow-
up. Nevertheless, a diﬀerence could be observed between
the high dose and control groups, which did exhibit com-
parable baseline values. is ﬁnding is consistent with
MRI observations and is in agreement with previous
reports that also investigated the role of cultured MSCs or
MSCs embedded in scaﬀolds in knee OA [23, 24, 42–44].
e required dose of MSCs to treat knee OA eﬃciently is
a topic of active research. Recently Jo etal.  performed
a pilot study comparing three doses of cultured adipose
tissue-derived MSCs (1×106, n=3; 50×106, n=3; and
100×106, n= 3). ey found a signiﬁcant reduction in
the VAS score only in the high dose group at 6months,
in spite of the small number of patients included. Since
results were better with the highest dose, they focused on
this in a second phase of the study 100×106 (n=9), with
promising results. Our ﬁndings also suggest that it is pref-
erable to administer 100×106 rather than 10×106 cells.
However, we have to bear in mind that, despite randomi-
zation, the OA degree at recruitment was more severe in
the patients who received only 10×106 cells, which may
obscure our interpretation of this result.
Page 8 of 9
Lamo‑Espinosa et al. J Transl Med (2016) 14:246
It is accepted that OA patients have a MSC deﬁcit that
leads to a degenerative process, and the number, invitro
proliferation and diﬀerentiation potential of BM-MSCs
present in the subchondral bone decreases with age and
OA grade [10, 11, 45]. However, we were able to obtain a
suﬃcient amount of BM-MSCs in osteoarthritis patients,
regardless of their age or grade of disease [46–49]. We
have not identiﬁed any problems during the process of
production of autologous BM-MSCs, achieving the num-
ber of autologous BM-MSCs proposed, even though the
mean age of patients was around 60years.
e present study is not exempt from limitations. First,
ethical issues prevented us from performing a double-blinded
trial. In order to minimize this inconvenience, subjective
clinical scores were contrasted with objective measures to
minimize bias. In addition, two independent radiologists
carried out the MRI analyses in a blinded manner. Second,
the relatively short duration of the study prevented us from
analyzing the eﬃciency of the treatments beyond 1 year
after the administration of the treatments. Finally, as antici-
pated, the severe initial condition of a portion of patients who
were going to be administered the low dose of cells may have
stopped these exerting more beneﬁcial eﬀects.
Our study shows that the single intraarticular injection
of invitro expanded autologous BM-MSCs together with
HA is a safe and feasible procedure that results in a clini-
cal and functional improvement of knee OA, especially
when 100×106 cells are administered. ese results pave
the way for a future phase III clinical trial.
Additional le1: Figure S1. Pattern of treatment administration. BM‑
MSCs (bottom right inset) were administered in two consecutive intraar‑
ticular injections with a 19 G needle using a lateral patellar approach.
10 × 106 or 100 × 106 cells were injected in 1.5 and 3 ml respectively and
subsequently 60 mg hyaluronic acid were administered in 4 ml. Patients
randomized to the control group received solely the second injection.
Additional le2: Figure S2. A–C, methacrylate patient positioner to
permit a correct caption of Rosenberg X‑ray projections. The X ray tube
is placed behind the patient, at the level of the knee and at an angle of
10° with respect to the horizontal in order to evaluate the knee articular
width. D, examples of the X‑ray images, obtained at baseline and 6 and
12 months afterwards, of the knees of three of the recruited patients are
shown. For each patient, images are comparable to each other, which
makes it possible to obtain a valid and comparable value of the articular
Additional le3: Table S1. VAS before administration of treatments and
3, 6 and 12 months afterwards.
Additional le4: Table S2. Goniometric measurements of the knee ﬂex‑
ion and extension ranges of motion before administration of treatments
and 3, 6 and 12 months afterwards.
Additional le5: Table S3. X‑ray measurement of the knee articular
interline before administration of treatments and 6 and 12 months
αMEM: alpha minimum essential medium; bFGF: ﬁbroblast growth factor;
BM‑MSCs: bone marrow mesenchymal stromal cells; GMP: good manufacture
practices; HA: hyaluronic acid; ISCT: International Society for Cellular Therapy;
K‑L: Kellgren and Lawrence scale; MRI: magnetic resonance imaging; MSCs:
mesenchymal stromal cells; OA: osteoarthritis; VAS: visual analogue scale;
WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index;
WORMS: Whole‑Organ Magnetic Resonance Imaging Score.
Study design: JML‑E, GM, JM‑NC, MCC, FP. Provision of study materials or
patients: JML‑E, GM, JFB, AV‑A, EMV, GS‑G, JRV‑N, EA, FP. Data collection and
assembly: JML‑E, GM, EA, JFB, JMN‑C, FG‑M, CS‑E, JMB, JD‑A. Obtaining of
funding: FG‑M, MCC and FP. Drafting manuscript: JML‑E, FG‑M, FP. JML‑E, FG‑M,
JMN‑C, FP take responsibility for the integrity of the data analysis. All authors
read and approved the ﬁnal manuscript.
1 Department of Orthopaedic Surgery and Traumatology, Clínica Universidad
de Navarra, Pamplona, Spain. 2 Department of Orthopaedic Surgery and Trau‑
matology, IBSAL‑Hospital Universitario de Salamanca, Salamanca, Spain.
3 TerCel (Spanish Cell Therapy Network, Spanish National Institute of Health
Carlos III), Madrid, Spain. 4 Cell Therapy Area, Clínica Universidad de Navarra,
Pamplona, Spain. 5 Navarra Institute for Health Research (IdiSNA), Pamplona,
Spain. 6 Division of Biostatistics, Research Support Service, Central Clinical
Trials Unit, Clínica Universidad de Navarra, Pamplona, Spain. 7 Department
of Preventive Medicine and Public Health, Medical School, University of Nav‑
arra, Pamplona, Spain. 8 Department of Radiology, Clínica Universidad de
Navarra, Pamplona, Spain. 9 Department of Rheumatology, Clínica Universidad
de Navarra, Pamplona, Spain. 10 Department of Hematology, IBSAL‑Hospital
Universitario de Salamanca, Salamanca, Spain. 11 Department of Hematology,
Clínica Universidad de Navarra, Avenida Pío XII 36, 31009 Pamplona, Navarra,
Spain. 12 Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla
y León, Castilla y León, Salamanca, Spain.
The authors declare that they have no competing interests.
Availability of data and materials
All the data presented is available upon request.
Ethics approval and consent to participate
All the procedures were approved by the Institutional Review Board of Navarra
and the Spanish Agency of Medicines and Medical Devices.
This work has been partially supported by Grants PI13/01633 (MINECO
through Instituto de Salud Carlos III to FG‑M) and RD12/0019/0017 (to MCC)
and RD12/0019/0031 (to FP) from Instituto de Salud Carlos III (red TerCel). EMV
is supported by Centro en Red de Medicina Regenerativa y Terapia Celular de
Castilla y León, Consejería de Sanidad, Junta de Castilla y León.
Received: 22 June 2016 Accepted: 2 August 2016
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