Regenerative Therapy 24 (2023) 147e153

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Regenerative Therapy
journal homepage: http://www.elsevier.com/locate/reth

Original Article

Multiple intra-articular injections with adipose-derived stem cells forknee osteoarthritis cause severe arthritis with anti-histone H2Bantibody production
Yuji Hosono a, Ayano Kuwasawa b, Eriko Toyoda c, d, Kotaro Nihei b, Shinji Sato a,
Masahiko Watanabe c, d, Masato Sato c, d, *a

Division of Rheumatology, Department of Internal Medicine, Tokai University School of Medicine, 143 Shimokasuya Isehara, Kanagawa, 259-1193 Japan
Saitama Cooperative Hospital, 1371 Kisoro, Kawaguchi, Saitama, 333-0831, Japan
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193 JapandCenter for Musculoskeletal Innovative Research and Advancement (C-MiRA), Tokai University Graduate School, 143 Shimokasuya, Isehara, Kanagawa,259-1193 Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 March 2023
Received in revised form
9 May 2023
Accepted 13 June 2023

Introduction: Osteoarthritis (OA) is the most common form of arthritis. OA results from the breakdown ofcartilage, which leads to deterioration of the entire joint and the connective tissue that holds the joint
together, and gradually and irreversibly worsens over time. Adipose-derived stem/stromal cells (ADSCs)
have been used in the treatment of knee OA. However, the safety and efﬁcacy of ADSC treatment of OA
remain unclear. In this study, we investigated the pathophysiology of severe knee arthritis that occurred
after ADSC treatment by screening for autoantibodies in synovial ﬂuid from patients who received ADSC
treatment.
Methods: Adult Japanese patients with OA who received ADSC treatment at Saitama Cooperative Hospital between June 2018 and October 2021 were enrolled. Antibodies (Abs) were screened using
immunoprecipitation (IPP) with [35S]-methionine-labeled HeLa cell extracts. The detected protein was
identiﬁed by liquid chromatography coupled with time-of-ﬂight mass spectrometry (MS) and ion trap
MS, and the corresponding proteins were conﬁrmed as autoantigens using immunoblotting. Ab titers
were measured using an enzyme-linked immunosorbent assay.
Results: A total of 113 patients received ADSC treatment, and 75% (85/113) received ADSC injection atleast twice with a 6-month interval between. No obvious abnormalities were observed in any patient
after their ﬁrst treatment; by contrast, 53% (45/85) of patients who received their second or third ADSC
injection showed severe knee arthritis. IPP detected a common anti-15 kDa Ab in synovial ﬂuid of 62% (8/13) of the samples analyzed from patients who showed severe arthritis. This Ab was not detected insynovial ﬂuid obtained from the same joints before treatment. The corresponding autoantigen was
identiﬁed as histone H2B. All available synovial samples from patients who tested positive for antihistone H2B Ab were newly positive after the treatment; that is, none had been positive for antihistone H2B Ab before treatment.
Conclusions: Multiple ADSC injections for OA induced severe arthritis in a high percentage of patients,
particularly after the second injection. Synovial ﬂuid from some patients with knee arthritis contained Ab
to histone H2B that appeared only after ADSC treatment. These ﬁndings provide new insights into thepathogenesis of ADSC treatment-induced severe arthritis.
© 2023, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Keywords:
Adipose-derived stem/stromal cells
Anti-histone H2B antibody
Osteoarthritis
Knee joint* Corresponding author. Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193 Japan
E-mail addresses: yuji.hosono25@gmail.com (Y. Hosono), ayanofanfare@hotmail.com (A. Kuwasawa), etoyoda@tsc.u-tokai.ac.jp (E. Toyoda), niheikotaro@yahoo.co.jp(K. Nihei), shinsjam@tokai-u.jp (S. Sato), masahiko@is.icc.u-tokai.ac.jp (M. Watanabe), sato-m@is.icc.u-tokai.ac.jp (M. Sato).
Peer review under responsibility of the Japanese Society for Regenerative Medicine.
https://doi.org/10.1016/j.reth.2023.06.007
2352-3204/© 2023, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

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Regenerative Therapy 24 (2023) 147e153

environment [13]. ADSCs are of interest for clinical use in cell-based
therapy, and favorable outcomes have been reported for their use in
the treatment of knee OA [14,15]. However, the mechanisms
responsible for the therapeutic effects of ADSCs have been only
partially investigated, and the long-term safety and side effects ofthis treatment remain unclear.
Recently, we found that some patients with OA who received
ADSC treatment presented with severe arthritis and fever. Therefore, we have investigated the pathophysiology of this apparent
severe arthritis that occurs after ADSC treatment using autoantibody screening of synovial ﬂuid obtained from patients who
received ADSC treatment.

Abbreviations
ADSCs
Adipose-derived stem/stromal cells
Abs
Antibodies
DILE
drug-induced lupus erythematosus
ELISA
Enzyme-linked immunosorbent assay
HHDNA
human histone H2A-H2B-DNA
IPP
immunoprecipitation
LCMS-IT-TOF a liquid chromatograph mass spectrometer thatcombines a high-performance liquid
chromatograph with an ion-trap (IT) mass
spectrometer and time-of-ﬂight (TOF) mass
spectrometerMSmass spectrometry
MSCs
multipotent stem cells
MS/MS
Tandem mass spectrometry
OA
Osteoarthritis (OA)
PAGE
polyacrylamide gel electrophoresis
PVDF
polyvinylidene ﬂuoride
SDS
sodium dodecyl sulfate
SLEsystemic lupus erythematosus

2. Materials and methods
2.1. Patients and synovial ﬂuid samples
Ethical approval for the study protocol was obtained by theinstitutional review board of Saitama Cooperative Hospital(approval number: 170304). Adult Japanese patients with OA of theknee who received ADSC treatment at Saitama Cooperative Hospital (Kawaguchi, Saitama, Japan) between June 2018 and October
2021 were enrolled. Clinical data were collected retrospectively
from clinical chart review for each patient. Severe arthritis wasdeﬁned as arthritis that is painful at rest, unresponsive to analgesics, and presents with difﬁculty walking. The experiments using
remaining samples were performed under the approval and guidance of the Clinical Research Review Committee of Tokai University
School of Medicine (approval number: 22R072) and review boardof Saitama Cooperative Hospital.

1. Introduction
Osteoarthritis (OA) is the most prevalent musculoskeletal disorder. OA induces joint pain and physical disability and increases
the risk of complications, sleep disturbances, reduced capability forexercising, lifting, and walking and are less capable of working [1].
The etiology of OA is multifactorial and includes many factors suchas age, sex, overweight, prior joint injury, medication, environmental factors, and genetics [2e4]. OA can cause progressive loss
and destruction of cartilage, sclerosis of the subchondral bone, andinﬂammation of the synovium, which eventually lead to continuous arthritis. Symptomatic OA may also impair the ability to bephysically active and to lose weight, which can lead to the vicious
cycle of weight gain and continued physical inactivity andincreased risk of associated conditions. Thus, treatment of OA is
critical to maintaining physical ability and improving healthy life
expectancy.
Traditionally, OA management includes nonpharmacologicaltreatments such as exercise, weight loss, and patient education, andpharmacological treatments including anti-inﬂammatory medications and intra-articular injections. These treatments are effective
for the early to intermediate phases of OA but are insufﬁcient for
progressive OA. Surgical options are considered for patients with
progressive OA, but their effects are often temporary, and OA continues to progress [5]. In addition, patients with OA often have risk
factors that can be contraindications for surgery, such as old age
and other conditions. Thus, novel interventions to restore degradedcartilage or decelerate OA disease progression are required.
The use of adipose-derived stem/stromal cells (ADSCs) has
recently been applied clinically for cartilage regeneration [6,7].
Since the discovery and characterization of multipotent stem cells(MSCs) in adipose tissue by Zuk et al. [8], ADSCs have been identiﬁed as a source of MSCs. MSCs are generally nontumorigenic andcan be isolated from many sites, including adipose tissue, bonemarrow, umbilical cord, dental pulp, endometrium, synovial ﬂuid,
muscle, and peripheral blood [9e11]. ADSCs can be easily and safely
harvested from abundant adipose depots with low risk to the patient [12]. These cells can secrete many kinds of cytokines andgrowth factors and recruit macrophages, which can play an
immunosuppressive role after exposure to an inﬂammatory

2.2. ADSC treatment
Adipose tissue was collected by liposuction from the knees ofpatients with OA. ADSCs were isolated from the knee joints of patients with OA undergoing aesthetic or prosthetic surgery at Saitama Cooperative Hospital, following a well-established protocol[12]. With the patient under local anesthesia, 20 ml of abdominal
fat tissue was collected in an operating room. The collected fat was
kept in normal saline at 4  C and mailed within 48 h to the cellprocessing center at CellSource Co., Ltd. (Shibuya, Tokyo, Japan).
The entire 20 ml adipose tissue sample for each patient wascultured for 2 weeks. The medium was replaced every other day
and, when they reached 70e80% conﬂuence, the cells were detached, plated, expanded, and cryopreserved until the day ofadministration, as described previously [16]. For injection, the
ADSCs were washed twice, and 15 million cells were diluted with
3 ml of saline and injected into the intra-articular space of the knee.
Some patients received this injection two or three times at 6month intervals.
2.3. Antibody (Ab) detection
Ab in synovial ﬂuid was screened by immunoprecipitation (IPP)
using radiolabeled HeLa cell extracts as previously described [17].
Brieﬂy, 1  107 HeLa cells were labeled with 9.25 MBq of [35S]methionine (PerkinElmer, Waltham, MA, USA) in 10 ml ofmethionine-free minimal essential medium and incubated at 37  C
for 18 h. The labeled cells were washed three times withphosphate-buffered saline (137 mmol/l NaCl, 8.1 mmol/l Na2HPO4,
2.68 mmol/l KCl, 1.47 mmol/l KH2PO4) and sonicated in IPP buffer(10 mM TriseHCl, 500 mM NaCl, 0.1% Nonidet P-40). The solublesupernatant in IPP buffer was collected by centrifugation at
10,000 g for 10 min. Two microliters of synovial ﬂuid was bound to
2 mg of Protein A CL-4B Sepharose beads (GE Healthcare, Uppsala,148

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Regenerative Therapy 24 (2023) 147e153

acetonitrile in 50 mM NH4HCO3 followed by 60% acetonitrile in
50 mM NH4HCO3, and the samples were dried using a SpeedVac
concentrator (Thermo Fisher Scientiﬁc, Waltham, MA, USA). For ingel digestion of proteins, the samples were incubated with a trypsin
solution at 37  C overnight, and the extracted trypsinized peptides
were eluted stepwise with 30%, 100%, and 0% of acetonitrile in
50 mM NH4HCO3. The samples were dried and then dissolved in
50 mM NH4HCO3. Tryptic peptides were separated by reversedphase liquid chromatography/MS using a Prominence nanoLC instrument (Shimadzu, Kyoto, Japan) for analytical separation on a
PicoFritTMBetaBasic C18 column (100 mm  75 mm; New Objective,
Woburn, MA, USA). MS was performed on an LCMS-IT-TOF instrument with argon gas for ion cooling and collision-induced dissociation experiments. MS/MS data were obtained in a datadependent manner. The Mascot search engine (Matrix Science,
Boston, MA, USA) was used for protein database searching. Proteins
with a statistically signiﬁcant MASCOT/Mowse score (>28), indicating identity or extensive homology (P < 0.05), were considered
for identiﬁcation.

Sweden) in IPP buffer for 2 h at room temperature under constantrotation. After washing four times with IPP buffer, IgG-coatedSepharose beads were mixed with the [35S]-methionine-labeled
HeLa cell extracts for 2 h at 4  C. After washing four times with IPP
buffer, the IgG-coated beads were resuspended in sodium dodecyl
sulfate (SDS) sample buffer and heated for 5 min at 95  C. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis(PAGE) and visualized by radiography.
2.4. Identiﬁcation of the autoantigen recognized by the anti-15 kDa
Ab
To identify the corresponding autoantigen, immunoprecipitatedpolypeptides from HeLa cells without [35S]-methionine labelingwere visualized by silver staining. The corresponding silver-stained
gel band at 15 kDa was excised and subjected to liquid chromatography coupled with time-of-ﬂight MS and ion trap MS (LCMS-ITTOF) analysis.
2.5. Immunoblotting

3. Results
Immunoblotting to conﬁrm the antigen identity was performed
according to a modiﬁed procedure detailed previously [17]. In
short, Recombinant Human Histone H2B protein (NBP3-07913,
Novus Biologicals, Centennial, CO, USA) was electrophoreticallyseparated by SDS-PAGE using a 4e15% Mini-PROTEAN® TGX™ gel(Bio-Rad, Hercules, CA, USA) and later transferred to a polyvinylidene ﬂuoride (PVDF) membrane. After transfer, the PVDF
membrane was stained with Ponceau S Staining Solution (BCLPSS-01, Beacle, Inc., Sakyo, Kyoto, Japan). After conﬁrming thetransferred protein, the PVDF membrane was washed, and eachlane was cut out and incubated with 5% nonfat dry milk in Trisbuffered saline with Tween 20 (TBST, pH8.0) for 1 h at room
temperature. The blots were washed with TBST three times for
5 min each and then incubated overnight at 4  C with primary Abs
contained in the synovial ﬂuid obtained from patients with OA,
serum from healthy controls, commercial anti-histone H2B Ab(GTX129434, GeneTex, Irvine, CA, USA), and anti-histone-Abpositive serum from a patient with systemic lupus erythematosus (SLE). The membranes were washed with TBST ﬁve times for
5 min each, incubated with the second Ab against human or rabbit
IgG (anti-human IgG AP conjugate, S3821 and anti-rabbit IgG AP
conjugate, S3731, Promega K$K., Tokyo, Japan). The blots were
washed ﬁve times with TBST and developed with BCIP/NBT Color
Development Substrate following the manufacturer's instructions(Promega S3771).

3.1. Severe arthritis induced by ADSC injection
A total of 113 patients with knee OA received ADSC treatment;
75% (85/113) had received ADSC injection twice or more at 6month intervals. No obvious abnormalities were observed in any
patient after their ﬁrst treatment. However, surprisingly, 53% (45/85) of patients who received their second ADSC injection showedjoint swelling, severe pain, and redness. Fever was noted along withsevere arthritis in 11% (9/85) of patients who received a second
ADSC injection. Considering the possibility that multiple ADSC
treatments had induced severe arthritis, we investigated thepathogenesis by screening for autoantibodies in synovial ﬂuid fromthe patients who showed severe arthritis.
3.2. Screening for anti-15 kDa Abs
Synovial ﬂuid was available both before and after the second or
third injections for eight of the 45 patients (18%) who showed severe arthritis after multiple ADSC injections. We used IPP with
[35S]-methionine-labeled HeLa cells to screen for autoantibodies insynovial ﬂuid obtained from these patients (Fig. 1). A common
15 kDa polypeptide was immunoprecipitated from the synovial
ﬂuid of 62% (8/13) of the samples analyzed (Fig. 1A, black arrow).
Interestingly, the 15 kDa polypeptide was not precipitated from thesynovial ﬂuid from any patient after the ﬁrst injection but was
immunoprecipitated from the synovial ﬂuid obtained from patients
after the second and third injections (Fig. 1A, pre: before treatment,
and post: after the second or third injection). Interestingly, none of
the synovial ﬂuid samples obtained from any patient after the ﬁrst
injection immunoprecipitated the 15 kDa polypeptide, but thoseobtained from patients who showed severe arthritis after the second and third injections did (Fig. 1A, pre: before treatment, andpost: after the second or third injection). It was not detected insamples from patients without severe arthritis.

2.6. Enzyme-linked immunosorbent assay (ELISA) to measure antihistone H2B Ab titer
Synovial ﬂuid samples were frozen and used for the measurement of anti-histone H2B Ab titer by ELISA using a Human Histone
H2A-H2B-DNA Autoantibody ELISA Kit (MyBioSource, MBS029740,
San Diego, CA, USA). The ELISA plate was read at 450 nm, and thecutoff value as an indicator of a negative response to anti-histone
H2A-H2B-DNA Ab was deﬁned as the mean plus 0.15 of theabsorbance of the negative controls according to the manufacturer's instructions.

3.3. Identiﬁcation of the autoantigen recognized by the anti-15 kDaAb

2.7. Tandem mass spectrometry (MS/MS) and protein identiﬁcation
Next, to identify the immunoprecipitated common 15 kDa
protein, synovial ﬂuid that had immunoprecipitated the 15 kDa
protein was coupled with Protein A CL-4B Sepharose beads andincubated with HeLa cell extracts without [35S]-methionine labeling. The polypeptides were then fractionated by SDS-PAGE and

Identiﬁcation of the protein in the gel spots was performed
using an LCMS-IT-TOF instrument (Shimadzu, Kyoto, Japan). The
pieces of interest were excised from the gel, and in-gel digestion ofproteins was performed. The gel pieces were washed with 30%
149

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Regenerative Therapy 24 (2023) 147e153

Fig. 1. IPP of polypeptides with patient synovial ﬂuid samples and silver staining. (A) Synovial ﬂuid from patients with OA contained the anti-15 kDa antibody. Using [35S]
methionine-labeled HeLa cell extracts, antigens were precipitated with the synovial ﬂuid from patients with OA (1e4) and serum from healthy control. HC; healthy control, A;
before treatment, B; post second injection. (B) HeLa cell extracts reacted with Patient No.4B synovial ﬂuid were fractionated on SDSepolyacrylamide gel and visualized by silver
staining.

before and after ADSC treatment were analyzed by ELISA (Table 1).
Interestingly, all of these samples were negative for anti-histone
H2B Ab before treatment and were newly positive only after
treatment. These results suggest that anti-histone H2B Ab did notexist in the synovial ﬂuid before ADSC treatment but appeared only
after multiple ADSC treatment.

visualized by silver staining (Fig. 1B). The band corresponding to
15 kDa was cut out and subjected to LCMS-IT-TOF analysis, and thisanalysis suggested histone H2B as the candidate corresponding
protein (Fig. 2).
3.4. Conﬁrmation of histone H2B as the corresponding autoantigen
to anti-15 kDa Ab

4. Discussion

To conﬁrm that histone H2B was recognized by the anti-15 kDa
Ab, immunoblotting using recombinant human histone H2B protein was performed. Histone H2B protein on the PVDF membranes
reacted with the commercial anti-histone H2B Ab, synovial ﬂuid
samples that immunoprecipitated the common 15 kDa protein, andanti-histone H2B-positive serum from a patient with SLE, whereas
samples without the anti-15 kDa Ab did not (Fig. 3). This conﬁrmedthat histone H2B was the autoantigen corresponding to the anti-15
kDa Ab.

This study reports some novel ﬁndings regarding ADSC treatment in patients with knee OA. First, we found that severe knee
arthritis was induced in a high percentage of patients after multiple
ADSC injections. Second, IPP detected anti-histone H2B Ab in synovial ﬂuids from the patients who showed severe knee arthritis
after treatment. Third, anti-histone H2B Ab appeared to be newly
induced after multiple ADSC treatment.
OA is the most common form of arthritis and is treated in many
ways including intra-articular injection. However, OA is generally
progressive and leads to irreversible destruction of cartilage,
which is a major cause of disability. Although the exact pathogenesis of knee OA is unclear, continuous inﬂammation
throughout the entire joint structure leads to the degradation of

3.5. Anti-histone H2B Ab titer before and after ADSC treatment
To identify when anti-histone H2B Ab appeared and to measurechanges in Ab titers, synovial samples that were available bothFig. 2. MASCOT search results. The silver-stained band corresponding to 15 kDa was excised and digested with trypsin. The peptide mixture was analyzed in duplicate with MALDITOF MS. MALDI-TOF spectra of peptide ion masses from the protein digest were used to predict the protein sequence. Mascot was used to compare the observed spectra with a
database of known proteins and determine the most likely matches. The results showed histone H2B as the candidate protein corresponding to the immunoprecipitated autoantigen. Protein sequence coverage was 8%.
150

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Regenerative Therapy 24 (2023) 147e153

Fig. 3. Immunoblot analysis of histone H2B protein as the corresponding autoantigen. (A) Ponceau S-stained PVDF membrane. Recombinant human histone H2B protein was
separated electrophoretically by SDS-PAGE using a 4e15% Mini-PROTEAN® TGXTM gel, transferred to a PVDF membrane, and stained with Ponceau S to conﬁrm protein transfer. (B)After cutting the each lane, the membranes were reacted with the anti-15 kDa-positive synovial ﬂuid, anti-15 kDa-negative synovial ﬂuid, commercial anti-histone H2B antibody,and anti-histone H2B-positive serum from a patient with SLE. Lane 1: molecular weight marker; lane 2: commercial anti-histone H2B antibody; lanes 3e6: anti-15 kDa-positive
synovial ﬂuid; lanes 7e9: anti-15 kDa-negative synovial ﬂuid; and lane 10: anti-histone H2B-Ab-positive serum from a patient with SLE.

Table 1
Patients who experienced knee pain and presence of anti-human histone H2A-H2B-DNA autoantibody (HHDNA) in their synovial ﬂuid before and after treatment.
Patient
numberAge(years)Sex

Num-berof ADSC
treat-ments

KL
grade

4
4
4
4
3
4
2
3
4
4
2
3

1

83

F

2

2

86

F

2

3
4

63
56

M
F

2
2

5a
6

76
52

F
F

3
2

7b
8c

64
66

F
F

2
3

Knee
joint

R
L
R
L
R
R
L
L
R
R
L
R

Presence of anti-HHDNA in synovial
ﬂuid before and after treatment

Volume of synovial
ﬂuid aspirate (mL)

BeforeAfter

BeforeAftere

e
e
e
e
e
e
e
e
e

þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ

5.8
1.0
8.0
6.0
15.0
8.5
17.0
7.2
12.0

12.0
2.0
8.0
6.0
14.0
1.0
14.0
9.2
35.0
2.0
6.8
12.2

e
e

7.0
4.8

Appea-rance ofsynovial
ﬂuid aspirate

Duration ofswelling, heat, and pain
after ADSC injection (days)

PYd
PY
PY
PY
PY
PY
PY
PY
PY
PY
PY
PY

5
7
1
4
30
2
1
10

a
Patient 5 received ADSC injection into the right knee in the ﬁrst treatment, into both knees in the second treatment, and into the left knee in the third treatment. Bothknees showed severe pain and swelling after the second ADSC injection.
b
Patient 7 received ADSC injection into the hip in the ﬁrst treatment.
cPatient 8 experienced knee pain after the second and third ADSC treatment. Pain was present for 10 days after the second, and severe pain for 1 week after the third.
d
PY: Pale yellow plasma-like.
eSynovial ﬂuid was aspirated 1 week after treatment for patient 1-4, 2 weeks for patient 5, 1 and 4 weeks for patient 6 and 4 weeks for patient 7 and 8.

by OA. However, in our study, 53% of the patients with knee OA
who were treated with ADSC injection showed symptoms of severe knee arthritis, especially after their second or third injection,and some patients developed fever. Although previous reports
have indicated the utility of ADSC treatment for patients with OA,
few studies have reported on the side effects of this procedure,
and its long-term safety remains unclear.
We found the identiﬁcation of anti-Histone H2B Ab production
with severe knee arthritis after ADSC injection, especially after
multiple injections. Of note is the high frequency of anti-histone
H2B Ab detected in the synovial ﬂuid from those patients who
showed symptoms of severe knee arthritis. The corresponding
autoantigen histone is a protein component found in the nucleosome and consists of ﬁve subunits: H1 (21 kDa), H2A (14.5 kDa),
H2B (13.7 kDa), H3 (15.3 kDa), and H4 (11.3 kDa). Each nucleosome
contains core histones that form a spool that wraps 145e147 bp of
DNA [27]. Autoantibodies to histones were ﬁrst reported in theserum from patients with SLE and were later detected in the serum

articular cartilage, formation of subchondral bone, and mild synovitis [18]. Synovial tissues from people with OA demonstrate theinﬁltration of CD4þ and CD68þ cells, formation of blood vessels,
and expression of vascular endothelial growth factor and intracellular adhesion molecule 1 [19]. The expression of cytokines
such as tumor necrosis factor a, interleukin 1 (IL-1), IL-6, and IL-17,
which activate CD68þ macrophages, is also highly upregulated injoints with OA [18e24].
The anti-inﬂammatory effects of ADSC treatment have beenreported recently. For example, the intra-articular deposition of
ADSCs inhibited thickening and activation of the synovial lining
layer and protected against joint destruction in an experimental
OA model involving synovial activation [25]. In addition, theprocess of acquisition of ADSCs from adipose tissue allows for thedevelopment of anti-inﬂammatory properties and the suppressionof inﬂammatory agents produced by fat [26]. Thus, ADSC treatment for OA is considered a potent and safe tool for managing
inﬂammation and protecting against the joint destruction caused
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Regenerative Therapy 24 (2023) 147e153

from patients with drug-induced lupus erythematosus (DILE)[28e30]. It was estimated that 24e95% of patients with SLE and67e100% of those with DILE are positive for anti-histone Ab [31].
Among the ﬁve subunits of histone H2B, histones H1 and H2B are
prominent autoantigens that induce the production of anti-histoneAbs [32], H2B is the corresponding consistent with the results ofour study.
Many drugs can induce DILE symptoms such as arthritis, fever,muscle pain, or skin lesions [30]. Procainamide and hydralazine are
known triggers of lupus-like symptoms and anti-histone H2B
autoimmunity. These symptoms are similar to those associatedwith SLE, although the autoantibodies differ between DILE and SLE.
For example, anti-Sm and anti-RNP Abs are often detected in serumfrom patients with SLE, whereas both are rare in serum from patients with DILE [30]. Although the pathogenic mechanisms remain
unclear, DILE is temporally related to continuous exposure to somemedications and resolves after discontinuation of the medication.
Arthritis is the most frequent, but often the only, clinical symptom
and occurs in 90% of patients with DILE. In our study, none of synovial ﬂuid samples from the patients who showed severe arthritis
with anti-histone H2B Ab was positive for anti-Sm or RNP Ab by IPP
screening. Moreover, none of the cases of severe knee arthritis was
induced by a single injection, and symptoms appeared only after
the second injection; that is, anti-histone H2B Ab appeared to be
newly induced only after multiple ADSC treatment. Given theclinical course and the appearance of anti-histone H2B Ab, severe
knee arthritis induced by multiple ADSC injections may be similar
to DILE and may reﬂect an ADSC-induced autoimmune response.
Although our present report differs from conventional reports
regarding safety, the present report is not immediately intended todeny or cast doubt on the efﬁcacy or safety of ADSC treatment. One
article indicates that 10% of patients who received single ADSC
injection had pain and swelling for 4 weeks following ADSC therapy, and due to the observed impact on their usual activities of dailyliving, it was categorized as a severe adverse event. Nevertheless,none of those in a group with double ADSC injections weredetected as a severe adverse event [33]. Sekiya et al. reported thatmultiple MSC injections for patients with progressive OA suppressed cartilage loss without a severe adverse event [34]. Anotherarticle indicated that 50%e60% of those in an MSC treatment groupexperienced inﬂammation and swelling after the injection procedure [35]. However, the same symptoms were also observed in acontrol group. These reports support the safety and efﬁcacy of
ADSCs and the possibility of adverse events being due to the courseof administration rather than ADSCs themselves. Because antihistone H2B Ab production was observed along with severe
arthritis, which suggests some type of immune response was
induced, further investigation is required to fully elucidate thispoint.

6. Conclusions
We investigated the pathophysiology of severe arthritis thatappeared after ADSC treatment for OA of the knee. Anti-histone
H2B Ab was detected in synovial ﬂuid samples from those patients who exhibited severe arthritis. Anti-histone H2B Ab production was induced after the second ADSC treatment. Theseﬁndings provide information that may be useful in the development of improved treatments for OA involving ADSCs.
Authors’ contributions
Conception and design: YH, AK, ET, KN, SS, and MS; dataacquisition: AK, ET, KN, and MS; data analysis and interpretation:
YH, AK, ET, KN, and MS; drafting of the manuscript: YH, ET, and MS;
and revision of the manuscript for important intellectual content:
YH, ET, MW and MS. All authors have read and approved the ﬁnal
manuscript.
Declarations of competing interest
The authors declare that they have no known competing
ﬁnancial interests or personal relationships that could haveappeared to inﬂuence the work reported in this paper.
Acknowledgements
We thank the Life Science Support Center, Tokai University
Medical Science College Ofﬁce for support for the a liquid chromatograph mass spectrometer that combines a high-performanceliquid chromatograph with an ion-trap (IT) mass spectrometerand time-of-ﬂight (TOF) mass spectrometer (LCMS-IT-TOF)
analysis.
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5. Limitations
This study has three main limitations. First, synovial ﬂuid was
not collected at the time of each ADSC treatment because we
could not predict that severe knee arthritis would occur in such ahigh percentage of patients after the second injection. Second,
control group was not set since ADSC treatment was in clinicalpractice. Third, the patients were not treated with glucocorticoids,
which is commonly used for patients with DILE, and we wereunable to conﬁrm whether the clinical course and pathophysiology of the symptoms in these patients were fully compatible
with those of DILE. Future prospective studies are needed toclarify the pathophysiology of severe arthritis induced by ADSC
treatment.
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