Biomedical Research 2017; 28 (4): 1654-1658 ISSN 0970-938X

www.biomedres.info

Efficacy of autologous adipose tissue-derived stem cells with extracellular

matrix and hyaluronic acid on human hip osteoarthritis.
Jaewoo Pak1,2,3, Jung Hun Lee1,4, Kwang Seung Park4, Sang Hee Lee4*
1Stems Medical Clinic, Chungdam-dong, Gangnam-gu, Seoul, Republic of Korea
2TPH (TEDA-TICH affiliated hospital), Third Ave, TEDA, Tianjin, China
3Life Science Institute, Komplek Permata Senayan Jl, Tentara Pelajar, Patal Senayan-Kebayoran Lama Jakarta,
Indonesia
4Department of Biological Sciences, National Leading Research Laboratory, Myongji University, Myongjiro, Yongin,
Gyeonggido, Republic of Korea

Abstract
The aim of this study is to demonstrate that percutaneous injections of autologous Adipose Tissue-
Derived Stem Cells (ASCs) and Extracellular Matrix (ECM) in the form of adipose Stromal Vascular
Fraction (SVF), along with Hyaluronic Acid (HA) and Platelet-Rich Plasma (PRP) activated by calcium
chloride could regenerate cartilage-like tissue in human hip Osteoarthritis (OA) patient. Autologous
lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterwards, the lipoaspirates
were homogenized to obtain ECM. This homogenized lipoaspirates were then mixed with collagenase
and incubated. The ASCs/ECM-containing SVF was mixed with calcium chloride-activated PRP plasma
and HA, and this ASCs mixture was injected into a hip of a patient with OA. Pre-treatment and post-
treatment MRI T2 views, functional rating index, range of motion, and pain score data were analysed.
The patient’s Magnetic Resonance Imaging (MRI) data showed significant positive changes. Cartilage-
like tissue regeneration was evident in MRI studies of the patient. Along with MRI evidence, the
measured physical therapy outcomes in terms of range of motion, subjective pain, and functional status,
all improved. These results indicate that percutaneous injection of the ASCs mixture is a promising
minimally invasive therapy for OA of human hip.

Keywords: Adipose tissue-derived stem cells, Extracellular matrix, Human cartilage regeneration, Osteoarthritis.
Accepted on September 14, 2016

Introduction the adipose tissue, the resultant cartilage regeneration capacity

may vary widely [7]. Among the many reasons, some of the
Adipose Tissue-Derived Stem Cells (ASCs) in the form of factors that may affect the result of cartilage regeneration by
Stromal Vascular Fraction (SVF) have been widely used in ASCs can be due to ASC viability, adherence, and growth.
Korea over decades by plastic surgeons as a semi-permanent
volume expander. In June 2009, Korean Food and Drug It is well recognized that ECM may work as a scaffold and
Administration (KFDA) had allowed adipose SVF to be used enhance stem cell adherence at the site of the cartilage lesion
as a medical procedure when obtained and processed within a [8]. Also, ECM has been shown to excrete various growth
same medical facility with minimal processing [1]. Adipose factors which may also enhance the survival and growth of
SVFs contain various components, including Mesenchymal stem cells following direct local injections [8].
Stem Cells (MSCs) and Extracellular Matrix (ECM) [2]. Such In this report, Platelet-Rich Plasma (PRP) was used as a source
MSCs are termed Adipose Tissue-Derived Stem Cells (ASCs) of growth factors and as a differentiating agent for the ASCs
and have shown to regenerate cartilage in vitro [3,4]. In 2011, that were administered locally. PRP contains various growth
Pak has showed that ASCs contained in the adipose SVF can factors including transforming growth factor-beta (TGF-beta).
regenerate cartilage-like tissue in human Osteoarthritis (OA) These growth factors in PRP have been shown to have positive
patients [5]. Since then, many studies have confirmed that effects on growth and differentiation of various cells including
autologous ASCs in human adipose SVF are potential agents chondrocyte formation [9-11]. Hyaluronic Acid (HA) and
capable of regenerating cartilage in OA patients [6]. Due to ECM were used as scaffolding materials [8,12]. Furthermore,
lack of standardization in autologous adipose tissue processing we present a Magnetic Resonance Imaging (MRI) evidence of
procedures and large variations in the numbers of ASCs within

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cartilage-like tissue regeneration in human hip OA patient Extracellular matrix (ECM) preparation
using the ASCs mixture (ASCs and ECM in the form of
adipose SVF along with HA and PRP activated by calcium In order to homogenize the lipoaspirates for even penetration
chloride). of collagenase and also to obtain additional ECM, the packed
adipose tissue was transferred back to two 60 ml Luer-Lock
syringes connected to a manual homogenizer/ECM extractor
Case Report (CPLTM, Medicamatch, Ansan, Korea), and were cut 20 times
The new rules and regulation by the KFDA has made it to mince the lipoaspirates.
possible to use adipose tissue as a source of ASCs in Korea [1].
Informed patient consent was obtained prior to the surgical Preparation of autologous adipose-derived stem cells
procedures. The approval and consent to report single cases (ASCs) mixture
were waived by the Myongji University Institutional Review
Board committee (HSC) for case report. Further, this clinical ASCs were extracted through the use of digestive enzymes
study was in compliance with the Declaration of Helsinki and (0.07% type 1 collagenase; Adilase, Worthington, Lakewood,
regulation guidelines of the KFDA. NJ, USA) and centrifugation (300 g for 5 min) to separate and
remove collagenase [3-5]. The total volume of the solution
containing ASCs and ECM was 8.5 ml from 50 ml of packed
Inclusion and exclusion criteria, and outcome
adipose tissue. While preparing the ASCs and ECM, 30 ml
endpoints autologous blood were drawn along with 2.5 ml anticoagulant
The inclusion criteria, exclusion criteria, and outcome citrate dextrose solution (0.8% citric acid, 0.22% sodium
endpoints are listed as follows. Inclusion criteria: MRI citrate, and 0.223% dextrose; Baxter Healthcare Corp., Marion,
evidence of the hip OA; orthopaedic evaluation that NC, USA). After centrifugation (300 g at room temperature for
determined that patient was a candidate for a Total Hip 15 min, then 1200 g at room temperature for 5 min using
Replacement (THR) surgery; males or females; 50 years of age supernatant), 4.4 ml of Platelet-Rich Plasma (PRP) along with
or over; unwillingness to proceed with THR; failure of the Buffy coat were obtained and 3% (w/v) calcium chloride
conservative management; and on-going disabling pain. (0.1 ml; Choongwae Pharmaceutical Co., Gyeonggido, Korea)
Exclusion criteria: active inflammatory or connective tissue was added to the mixture to activate PRP. 0.5% (w/v)
disease thought to impact pain condition (i.e., lupus, hyaluronic acid (2 ml; Huons, Chungbuk, Korea) was added as
rheumatoid arthritis, fibromyalgia); active endocrine disorder a scaffold to this mixture. The ASCs mixture comprised of
that might impact pain condition (i.e., hypothyroidism, separated ASCs and ECM, together with activated PRP and
diabetes); active neurologic disorder that might impact pain Hyaluronic Acid (HA).
condition (i.e., peripheral neuropathy, multiple sclerosis);
active cardiac disease; and active pulmonary disease requiring ASCs mixture-based treatment
medications. Outcome endpoints: will be studied using pain
After the hip was cleaned with 5% povidone-iodine
score, physical therapy and MRI both pre- and post- treatment.
(Choongwae Pharmaceutical Co., Seoul, Korea) and draped in
The pain score and physical therapy will involve Visual
a sterile fashion, the injection site was anesthetized with 0.25%
Analog Scale (VAS), Functional Rating Index (FRI), and
ropivacaine (Huons, Chungbuk, Korea) superficially outside of
Range of Motion (ROM) that were determined as previously
joint capsule and diluted 0.125% lidocaine (Daehan
described [13,14].
Pharmaceutical Co., Gyeonggido, Korea) was used to
anesthetize inside the joint capsule. On the same day of
Restriction of medications liposuction, the ASCs mixture (14 ml) was injected into the hip
Patient was restricted from taking steroids, aspirin, Non- joint via anterior approach using a 90 mm, 18 gauge spinal
Steroidal Anti-Inflammatory Drugs (NSAIDs), and Asian needle under an ultrasound guidance. The patient was, then,
herbal medications for one week prior to the procedure. instructed to remain still for 60 minutes to allow settlement of
the ASCs mixture at the site of lesion [5,6]. Upon discharge
Liposuction from the clinic, the patient was instructed to exercise minimal
activities for one week. The patient returned for three
In the operating room, approximately 50 ml of packed adipose additional injections of PRP activated by calcium chloride at
tissue were obtained by liposuction of the subcutaneous layer weekly intervals for three weeks.
of the lower abdominal area using manual techniques [5]. The
adipose tissue was treated with collagenase (0.07% type 1, at Patient and treatment outcome
37°C for 40 min; Adilase, Worthington, Lakewood, NJ, USA)
and the Stromal Vascular Fraction (SVF) containing ASCs was The patient is a 50-year-old Korean female with bilateral hip
separated from the lipoaspirates by a fat stem cell isolator osteoarthritis. The patient had the chronic bilateral hip pain
(CPLTM, Medicamatch, Ansan, Korea). over 7 years that gradually increased. The patient was
apparently better with use of chronic NSAID and occasional
injections of steroids and HA. However, approximately one
year prior to the office visit, the patient’s symptoms

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Efficacy of autologous adipose tissue-derived stem cells with extracellular matrix and hyaluronic acid on human hip
osteoarthritis
deteriorated. The patient underwent arthroscopic debridement/
lavage but was not completely relieved of pain. The patient
noticed worsening of the pain after the procedure and was
offered THR surgery by the orthopaedic surgeon. However, the
patient was reluctant to go through THR surgery due to
potential side effects. At the time of initial evaluation, the
patient reported severe pain (VAS score: 9; Figure 1A and
could not walk well without an assistant device. ROM (Figure
1B) was decreased. A pre-treatment MRI demonstrated joint
space narrowing, irregular cortical margin with diffuse bone
marrow oedema (Figures 2A, 2C, 2E, and 2G).
This patient went through the identical procedure of receiving
autologous ASCs mixture as described in the Experimental
Methods section. After the 2nd week of ASCs mixture
injection, the patient’s pain improved over 30% and the ROM
improved more than 20% (Figures 1A and 1B). By the 4th
week, pain was relieved by more than 50% and ROM
improved over 30% (Figures 1A and 1B). When the patient
returned at 20 weeks after the injection, pain was relieved by
more than 70% and ROM was improved by 50% (Figures 1A
and 1B), and cartilage-like tissue regeneration was evident on
the MRI (Figures 2B, 2D, 2F, and 2H).

Figure 2. MRI sagittal (A, B, C, and D) and coronal (E, F, G, and H)

sequential T2 views of the hip from the patient #4. Pre-treatment MRI
scans (A (sequential image: 9/20), C (10/20), E (7/20), and G (8/20))
show cartilage lesions (arrows). Post-treatment MRI scans at 20
weeks (B (9/20), D (10/20), F (7/20), and H (8/20)) indicate
cartilage-like tissue regeneration (arrowhead) that has been repaired
by ASCs mixture-based treatment.

Discussion
This clinical case report provides clear MRI evidence of
cartilage-like tissue regeneration in OA of human hip by using
the ASCs mixture. Based on MRI features, the newly formed
tissue resembled that of cartilage; however, the true nature of
the newly formed tissue cannot be confirmed without a biopsy.
While cartilage regeneration in human knees using ASCs in the
form of SVF have been shown in numerous human patients,
this case report is the very first study suggesting possibly a
better improvement in current strategy of cartilage regeneration
by employing the ASCs mixture in human hip. In addition to
the MRI evidence, the patient’s symptoms and signs improved
as early as two weeks than previously reported by Pak [5]. The
patient reported symptom improvement by the second week of
Figure 1. Outcome of pain measurements (A) and range of motion (B) the treatment. The patient's reported improvement in symptoms
from the patient. Error bars indicate standard deviations. may probably be due to the presence of ECM and its factors in
addition to ASCs.

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ECM has been shown to work as a scaffold and shown to injection of stem cell therapy with the ASCs mixture may
excrete various growth factors [8]. Thus, ECM can potentially therefore provide an alternative to current treatment strategy
improve the symptoms earlier by enhancing the cell adherence for the treatment of hip osteoarthritis.
to the lesion. After the adherence, it is possible that the various
growth factors released by the ECM can stimulate the adhered Conclusion
cells and surrounding tissue to grow. Therefore, the patient
noticed the early clinical improvements. As shown previously, In the present case, percutaneous injections of autologous
the patient did not report 100% resolution of their symptoms. ASCs mixture to hip OA provided significant relief from
This may be due to the fact that OA is a disease of the whole earlier symptoms. All clinical criteria of FRI, VAS score, and
joint and not just cartilage. Thus, with improved cartilage ROM improved on the patient, along with significant MRI
regeneration, the patient may need to strengthen the related changes. Although no biopsy of the regenerated tissue was
tendons, ligaments, and muscles to further improve already performed, the tissue can be estimated to be cartilage-like in
improved joint conditions. nature by comparing with the surrounding cartilage and other
tissues. Although further studies are necessary to verify this
Cartilage regeneration with adipose SVF depends on numerous new procedure, percutaneous injections of autologous ASCs
factors: number of stem cells and their viability, adherence, mixture present a promising, minimally invasive option of
growth and differentiation of the injected ASCs. The number treating OA of human hip by regenerating cartilage-like tissue.
of stem cells obtained from each individual patient is very
much influenced by collagenase enzyme used. Stem cells exist Conflict of Interests
within the ECM of the adipose tissue and by breaking down
the ECM with collagenase; stem cells are released [3,4]. The authors declare that they have no competing interests.
However, use of collagenase at high concentrations is
detrimental to the stem cell viability, and lesser concentrations Acknowledgments
are not effective in releasing stem cells from the matrix [15].
Therefore, standardizing with correct mixture and dosage of The author acknowledges the support from the staff of Stems
the collagenase is very important. Standardization of the Medical Clinic. This work was supported by research grants
collagenase is further complicated by individual patient from the National Research Foundation of Korea (NRF)
variations. The number of stem cells that can be obtained from funded by the Ministry of Science, ICT and Future Planning
each individual patient varies greatly [7]. Aging and the degree (No. 2011-0027928 and No. 2016R1C1B2010308) and Marine
of obesity, for example, may affect the texture of subcutaneous Biotechnology Program (20150581, Development of
tissue of each individual patient. Thus, collagenase may have Technology for Biohydrogen Production using
different effects on each individual patient’s adipose tissue Hyperthermophilic Archaea) Funded by Ministry of Oceans
[16], and standardizing the correct amount of collagenase to be and Fisheries in Republic of Korea.
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