Safety and efficacy of allogenic placental mesenchymal stem cells for treating knee osteoarthritis: a pilot study

ARTICLE IN PRESS Cytotherapy, 2018; 00: 1 10

Safety and efficacy of allogenic placental mesenchymal stem D1X X cells for treating knee osteoarthritis: a pilot study

D2X XSHAYESTEH KHALIFEH SOLTANID3X1X , D4X XBIJAN FOROGHD5X1X , D6X XNASER AHMADBEIGID7X2X , D8X XHOMAYOUN HADIZADEH KHARAZID9X3X , D10X XKHADIJEH FALLAHZADEHD1X2X , D12X XLADAN KASHANID13X4X , 19X X D14X XMASOUMEH KARAMID515X X , D16X XYADOLLAH KHEYROLLAHD17X1X & D18X XMOHAMMAD VASEID2,6 1

Physical Medicine and Rehabilitation, Neuromusculoskeletal Research Center, Iran University of Medical Sciences, Tehran, Iran,2Cell-based Therapies Research Center, Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran,3Department of Diagnostic Radiology, Iran University of Medical Sciences, Tehran, Iran,4Department of Obstetrics and Gynecology, Arash Hospital, Tehran University of Medical Sciences, Tehran, Iran,5Department of Biochemistry, School of Medicine, AJA University of Medical Sciences, Tehran, Iran, and 6 Department of Pathology and Cell-based Therapies Research Center, Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran Abstract Objective: Knee osteoarthritis (OA) is a common skeletal impairment that can cause many limitations in normal life activities. Stem cell therapy has been studied for decades for its regenerative potency in various diseases. We investigated the safety and efficacy of intra-articular injection of placental mesenchymal stem D20X X cells (MSCs) in knee OA healing. Methods: In this doubleblind, placebo-controlled clinical trial, 20 patients with symptomatic knee OA were randomly divided into two groups to receive intra-articular injection of either 0.5 0.6 £ 108 allogenic placenta-derived MSCs or normal saline. The visual analogue scale, Knee OA Outcome Score (KOOS) questionnaire, knee flexion range of motion (ROM) and magnetic resonance arthrography were evaluated for 24 weeks post-treatment. Blood laboratory tests were performed before and 2 weeks after treatment. Results: Four patients in the MSC group showed mild effusion and increased local pain, which resolved safely within 48 72 h. In 2 weeks post-injection there was no serious adverse effect and all of the laboratory test results were unchanged. Early after treatment, there was a significant knee ROM improvement and pain reduction (effect size, 1.4). Significant improvements were seen in quality of life, activity of daily living, sport/recreational activity and decreased OA symptoms in the MSC-injected group until 8 weeks (P < 0.05). These clinical improvements were also noted in 24 weeks post-treatment but were not statistically significant. Chondral thickness was improved in about 10% of the total knee joint area in the intervention group in 24 weeks (effect size, 0.3). There was no significant healing in the medial/lateral meniscus or anterior cruciate ligament. There was no internal organ impairment at 24 weeks follow-up. Conclusion: Single intra-articular allogenic placental MSC injection in knee OA is safe and can result in clinical improvements in 24 weeks follow-up. Trial registration number: IRCT2015101823298N.

Key Words: mesenchymal stem cell, osteoarthritis, placenta

Introduction As life expectancy increases, age-related diseases will become a serious concern. According to the World Health Organization (WHO) report 2002, osteoarthritis (OA) is the fourth leading cause of disability worldwide with a burden mostly attributed to arthritis of hips and knees [1]. According to multiple resources including the Framingham Osteoarthritis Study, National Health and

Nutrition Examination Survey (NHANES) III and the Johnston County Osteoarthritis Project, 26.9 million people aged >25 years in the United States have experienced at least one form of OA up until 2005 [2]. Knee pain has a high prevalence in the Asian region [3] and is the most common musculoskeletal symptom in Iran [4,5]. OA is a progressive joint degenerative disorder that involves all structures of a joint, such as hyaline

Correspondence: Mohammad Vasei, MD, Cell-based Therapies Research Center, Digestive Diseases Research Institute, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran. Co-Correspondence: Bijan Forogh, MD, Physical Medicine and Rehabilitation, Neuromusculoskeletal Research Center, Iran University of Medical Sciences, Tehran, Iran. E-mails: [email protected], [email protected] (Received 15 August 2018; accepted 4 November 2018) ISSN 1465-3249 Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2018.11.003

ARTICLE IN PRESS 2 S. KHALIFEH SOLTANI et al. cartilage, subchondral bone and synovium, and can results in subchondral sclerosis, cyst or osteophyte formation and synovitis. All joints of the human body, especially the weight-bearing joints, can be affected in a multifactorial background, including genetics and environmental factors [6]. Recommended OA treatments based on American College of Rheumatology guidelines include both non-pharmacological (activity modification, patient education, physical therapy modalities, exercises, assistive devices, weight loss, splints) and pharmacological modalities (acetaminophen, oral and topical nonsteroidal anti-inflammatory drugs, tramadol, intra-articular corticosteroid injections) [7]. Despite the substantial prevalence of OA, a 2-year follow-up in comparison with placebo reported no approved medical treatments such as glucosamine, chondroitin sulphate, combinations thereof and celecoxib for reversing cartilage degeneration [8]. The conventional treatment is considered mainly to impede the disease process and minimize disability and pain, but not to regenerate the joint structures. Unsuccessful medical treatment may lead to surgical interventions as standard final approaches with considerable side effects, such as mortality [9], persistent postsurgical pain [10] and patient dissatisfaction [11]. Moreover, arthroscopic surgery for moderate to severe knee OA provides no additional benefit to optimized physical and medical therapies [12]. Cell therapy for treating OA has been studied for about two decades. Although the efficacy of autologous chondrocyte transplantation in cartilage defect reconstruction has been proven, efficacies remain questionable due to reported donor site morbidity, downregulation of chondrocytes with fibrocartilage formation and not being fully evaluated in end-stage OA [13]. The important practical considerations for clinical application of mesenchymal stem 1X2D X cells (MSCs) that may alter their regenerative potential are as follows: (i) cell source (autologous/allogenic), (ii) dose (from thousands to millions of MSCs), (iii) delivery system (arthroscopic or surgical MSC scaffold transplantation/intra-articular MSC injection), (iv) number of injections and (v) MSC combined injection with co-stimulators (platelet-rich plasma [PRP], hyaluronic acid [HA] or growth factors) [13,14]. MSCs can be harvested from different tissues of the human body (e.g., bone marrow [BM], adipose tissue [AT] and placental/umbilical cord tissue). Safe clinical applications of MSCs have been reported in non-hematologic [15], and hematologic disorders [16]. MSCs have remarkable potential for differentiating into chondral, osseous and AT [17] and have been widely studied for treating degenerative joint diseases [14]. Given the comparable regenerative potential and hypo-immunogenicity of

placental/umbilical cord blood MSCs as compared with BM- or AT-derived MSCs, placental/umbilical cord blood MSCs stand to become more accessible and noninvasive MSC sources [13,18 21]. Many published or ongoing studies on the safety and efficacy of AT- or BM-derived stem cells for treating mild to severe OA [14] report their safety [22,23] and variable promising therapeutic potential in knee OA [24,25]. The present study is one of the first reports of placenta-derived MSCs (PLMSCs) as an allogeneic source for treating knee OA. We designed this pilot study to assess the safety of single intra-articular injection of allogeneic PLMSCs and to obtain preliminary data of their therapeutic value in 10 patients with grade 2 4 Kellgren Lawrence (KL) knee OA.

Methods Patients Twenty patients with knee OA (grades 2 4 based on the KD2X XL criteria in knee standing anteroposterior and lateral radiographs were enrolled from November 2015 to December 2016. The study was registered in the Iranian registry of clinical trials (http://www.irct. ir) after being approved by the Ethics Committee of the Iran University of Medical Sciences. All participants signed an informed consent form before participation. Patients in both groups were allowed to use acetaminophen to alleviate pain if needed. The patients’ routine activities of daily living (ADL) were continued early after intervention and only heavy activities or prolonged walking were restricted for 1week post-injection. Donor-informed consent forms were obtained from healthy women having normal vaginal delivery. Donor eligibility and suitability was assessed referring to the national guidelines for cell manufacturing [26]. Physical examinations and past medical histories were checked to determine whether the patients met the inclusion criteria; the exclusion criteria are listed in Table 1. Blood examinations before and 2 weeks after the treatment were carried out for complete blood count, erythrocyte sedimentation rate, Creactive protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen, creatinine, urine analysis, rheumatoid factor, fasting blood sugar, glycated hemoglobin, total and direct bilirubin, triglycerides, cholesterol, high-density lipoprotein cholesterol, calcium, parathyroid hormone, thyroid-stimulating hormone and vitamin D. Knee standing Anteroposterior/Lateral (Ap/Lat) radiograph was analyzed to determine OA grade and knee varus/valgus degrees. The body mass index (BMI) was also calculated. Knee range of motion

ARTICLE IN PRESS Allogenic stem cell therapy in knee osteoarthritis

used to evaluate the intra-articular structures before and 24 weeks after treatment (Figure 1).

Table I. Exclusion criteria. Age <35 or >75 y Any acute or chronic infection Visible knee deformity (varus >10˚; valgus >20˚) Pregnant or lactating women Any sort of neoplasia BMI >35 Conditions along with impaired immune system Any inflammation in the joints or secondary OA Intra-articular injections during the last 3 mo History of knee surgery Kidney malfunction (creatinine >2.0 mg/dL) Liver malfunction (bilirubin >2.0 mg/dL; AST and ALT >100 IU/L) Uncontrolled diabetes mellitus

Cell production from placenta The donors of placenta were selected form full-term healthy mothers who had normal vaginal delivery without complication. All donors signed an informed consent form that had been prepared for the clinical trial and approved by the National Institute for Medical Research Development (NIMAD)D23X X ethics committee. The individual donor blood samples were tested for reactive transmissible infectious agents such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and cytomegalovirus (CMV). During MSC expansion from the placenta, routine microbial and fungal cultures were performed to detect possible contamination in the final products. In addition, the cultivated cells were tested to detect mycoplasma species during cell culture. Karyotyping was performed to identify possible numerical or structural chromosomal

BMI, body mass index; AST, aspartate aminotransferase; ALT. alanine aminotransferase.

(ROM) determined with a standard goniometer, visual analogue scale (VAS) and Knee OA Outcome Score (KOOS) questionnaire were evaluated before injection and in 2, 8 and 24 weeks after treatment. Knee magnetic resonance arthrography (MRA) was

66 patients Assessed for eligibility

44 were excluded: 20 did not meet the criteria

24 refused to participate

20 were randomized: 10 intervention group: (Received intra-articular MSC injection)

10 control group: (Received intra-articular saline injection)

20 completed follow-up and analysis

Baseline MRA, lab, and physical evaluation

3

Intra-articular injection

Day 1 and 2 followup: Phone call

Week 2 follow up-visit and lab evaluation

Week 8 follow-up: Visit

Figure 1. Study flow diagram.

Week 24 followup: Visit and second MRA evaluation

ARTICLE IN PRESS 4 S. KHALIFEH SOLTANI et al. abnormalities and the cultivated cells were discarded if there was any karyotype abnormality. PLMSC extraction and harvesting All procedures were performed in a Good Manufacturing Practice (GMP) facility (clean room) according to current GMP guidelines. Placenta (3 4 g) was selected picked, rinsed and minced into minute pieces. The minced tissue was washed three times with 9% sodium chloride solution to remove the remaining blood, before being incubated with 1 mg/mL GMP-grade collagenase NB6 (SERVA Electrophoresis GmbH) at 37˚C for 3 h, with shaking every 30 min. Then, 9% sodium chloride solution was added and the mixture was shaken and centrifuged. The supernatant was discarded and the cell pellet was cultivated in MSC complete medium containing Dulbecco’s Modified Eagle’s Medium (ATOCEL) supplemented with 10% pharmaceuticalgrade Australian-origin fetal bovine serum (ATOCEL). Primary cultures were maintained for 1 week in a 37˚C humidified 5% CO2 incubator in small digested residues; non-adherent cells were removed by changing the culture medium. New medium was added twice weekly. Upon approximately 80% confluence, adherent MSCs were passaged via animal origin free TrypLE Express enzyme to reach a sufficient number of MSCs for further clinical applications.

0.5 mmol/L 3-isobutyl-1-methylxanthine (SigmaAldrich) for 21 days. Osteogenesis was carried out using osteogenic medium containing 10 mmol/L b-glycerol-phosphate (Merck), 50 mg/mL ascorbic acid bi-phosphate and 100 nmol/L dexamethasone (Sigma-Aldrich). After 21 days of differentiation, oil red O and alizarin red staining confirmed the ability of PLMSCs to differentiate into adipocytes and osteoblasts (Supplementary Figure 2).

Cell preparation for injection Each donor placenta was used for two to three patients. Average passage of cells was 12 passages. The cells were injected via 10-mL syringe. The MSC group received intra-articular injection of MSCs (10 mL; 0.5 0.6 £ 108). This concentration was chosen considering the average amount of cells that had been used in the previous reported studies on MSC injection for knee OA trials [27 30]. Twenty patients with symptomatic knee OA were randomly put into a group (A or B), which was defined by a statistician using a random table (n = 10 in each group) to receive intra-articular injection of either 10 mL of placenta-derived MSCs (A group) or 10 mL of normal saline (B group).

Cell surface marker phenotyping The PLMSCs were stained with monoclonal antibodies against the cell surface markers CD105, CD73, CD90, CD31, CD34 and CD45 (eBioscience) for immunophenotyping. PLMSCs were harvested with TrypLE Express enzyme and suspended in 3% bovine serum albumin (SigmaAldrich) in phosphate-buffered saline (Biowest). Cells were stained for 1 h with the above-mentioned antibodies and isotype control antibodies and were analyzed using FACSCalibur (BD Biosciences). Flow cytometry analysis of the PLMSC immunophenotype revealed that the sub- cultured cells were positive for the MSC surface markers CD73, CD90 and CD105 and were negative for CD34, CD45 and CD31 (Supplementary Figure 1). Differentiation studies Primary PLMSC cultures were composed of heterogeneous populations with fibroblastic and epithelioid cells. Cell colonies were subcultured until becoming confluent in an average of 14 days, with disappearance of the epithelioid cell population. We induced PLMSC adipogenesis using induction medium containing 250 nmol/L dexamethasone and

Figure 2. (A, B) Chondrolysis and marginal spur due to OA is evident in the patellofemoral joint. The chondral thickness at the lateral side of the patella is about 2.7 mm; follow-up imaging shows that it is 3.5 mm without definite changes in the marginal spur. The signal intensity of cartilage before injection is heterogeneous; follow-up MRA shows markedly decreased heterogeneity. (C, D) Pre- and post-injection MRA of a patient in the control group showing that chondral thickness, signal intensity and spur formation are not significantly different.

ARTICLE IN PRESS Allogenic stem cell therapy in knee osteoarthritis We tried to accomplish the study blind to the patients, the interventionist physician, assessors and the final analyst. Only the cell therapy center was aware of the injection type (MSCs or normal saline), which was asked by the interventionist for type A or B injection fluid for each patient. Because the color of the cell product was slightly different from that of the placebo solution, to assure blinding of the treatment, the barrel of the all injection syringes had been completely covered. The injection substances that were prepared in the cell production unit were specified with a given code and were sent to the clinical section for being injected. Follow-up visits and data collection as well as MRA evaluations all were done blindly by the physician and the interpreter radiologist. The analyst was also blind to the groups that were assigned as group 1 and 2 in the presented data. Radiological imaging We performed MRA using a Siemens Avanto Fit 1.5T system. Gadolinium (20 mL; 0.6%, DOTAREM) was injected in sterile conditions by a lateral parapatellar approach. The images were reviewed in coronal, sagittal and axial views (in MarcoPacs software), and then interpreted by an expert radiologist with 25 years of experience who had been blinded to the treatment groups. The cartilage thickness was described in millimeters at 14 sites (Table 2); the minimum and maximum thickness was determined (totaling 28 points of thickness evaluated per patient), and presence (yes/no) of synovial hypertrophy in sagittal views; spur in coronal and sagittal views and erosion in coronal, sagittal and axial views

5

were compared with follow-up matched MRA images to determine whether the defects had progressed, regressed or remained stable. The medial and lateral meniscus was evaluated using the Crues and Stroller classification (including horizontal, longitudinal, radial, root, complex, displaced, buckethandle tear patterns) [31]. Anterior cruciate ligament (ACL) injuries were described qualitatively using a five-point grading system (intact, low, intermediate, high-grade tear and degenerative) in baseline MRA images, followed by progression, regression or stable grading at the 24-week MRA [32].

Data analysis The KOOS questionnaire, VAS, ROM and MRA results were analyzed in SPSS 22 blinded to the interventions. P  0.05 was regarded as statistical significance.

Results Patients Based on Kellgren-Lawrence (KL) criteria, 18 patients had grade 2 and 3 and two patients had grade 4 knee OA. There were no significant differences between the two groups regarding age (P = 0.730), gender (P = 0.736), BMI (P = 0.716) and knee OA grading (P = 0.752). Supplementary Table 1 presents the results of the investigations of the variables and demographic characteristics.

Safety Table II. The 14 MRA evaluation sites. 1 SLP 2 MLP 3 ILP 4 SMP 5 MMP 6 IMP 7 MFF 8 LFF 9 LCN 10 LCW 11 MCN 12 MCW 13 TCM 14 TCL

Superior lateral patella Middle lateral patella Inferior lateral patella Superior medial patella Middle medial patella Inferior medial patella Medial femoral facet Lateral femoral facet Lateral compartment, non weight-bearing Lateral compartment, weight-bearing Medial compartment, non weight-bearing Medial compartment, weight-bearing Tibial compartment, medial Tibial compartment, lateral

Max Max Max Max Max Max Max Max Max

Min Min Min Min Min Min Min Min Min

Max Min Max Min Max Min Max Min Max Min

Fourteen different sites of chondral thickness measurement in minimum and maximum at each site; 28 sites in total. Max, maximum; Min, minimum.

Four patients in the MSC group had increased local pain and mild effusion. Their symptoms were mild and self-limited within 48 72 h. Re-examination at 2 weeks after treatment showed that the laboratory parameters all were unchanged. In the 24-week clinical and radiological follow-up, there was no ectopic mass formation or any other clinical adverse effects.

VAS The mean pre-intervention VAS values of the MSC and control groups were both 6.90, and at the 2-week, 8-week and 24-week follow-ups were 4.40 versus 4.40, 4.60 versus 4.20 and 5.10 versus 3.30, respectively. The group-time interaction effect was not significant (P = 0.401), indicating that the changes in both groups were not different in these intervals and that there were no impacts of intervention on patient pain.

ARTICLE IN PRESS 6 S. KHALIFEH SOLTANI et al. The within-group comparisons revealed no significant difference in the pain criterion between the two treatment groups. ROM The mean pre-intervention ROM of the MSC and control groups was 81.00˚ and 111.80˚, respectively, which was significantly different (P = 0.002); the values at the 2-week, 8-week and 24-week follow-ups were 86.70˚ versus 117.30˚, 113.10˚ versus 121.60˚ and 119.20˚ versus 127.10˚, respectively. Because the base values of the groups were significantly different, they were used as covariates in the analyses (Supplementary Table 2). The interaction effect of group-time was significant (P = 0.044), indicating that the changes in both groups were different in these intervals. The difference was related to the comparison of the 8-week and 24-week follow-ups (P < 0.05); no significant differences were observed between the two groups within other intervals. Within-group comparisons revealed significant differences in the MSC group between pre-intervention and 8-week follow-up (P = 0.003), pre-intervention and 24-week follow-up (P = 0.002), 2-week and 8week follow-up (P = 0.004), 2-week and 24-week follow-up (P = 0.003) and 8-week and 24-week follow-up (P = 0.032). The maximum effect size calculated using Cohen’s d method (small, 0.2; medium, 0.5; large, 0.8; very large, 1.3) is between preintervention and the 24-week follow-up comparison, which is 1.4. The results indicate that the improvement in knee joint ROM in the MSC group was significant between the 2-week and 24-week follow-up. Improvement in ROM in the MSC group was also significant between all interval pairs except for the first and second visits (before and 2 weeks after treatment). In the control group, the improvement in joint ROM was not significant in all interval pairs. KOOS questionnaire Analysis of the KOOS questionnaire revealed significant improvement in ADL and symptom scores from the MSC injection timepoint until 8 weeks later. The MSC group also showed significant improvement in quality of life (QOL) and pain reduction between the 2-week and 8-week follow-up, and in sport and recreation (S/R) factors between the 2-week and 24-week follow-up. Within-group comparisons of the KOOS questionnaire in the MSC group were as follows. (i) QOL score improvement was significant between preinjection and 8-week follow-up (P = 0.028; effect

size, 0.9). (ii) ADL score improvement was significant between pre-injection and 2-week follow-up (P = 0.009) and pre-injection and 8-week follow-up (P = 0.001; effect size, 1.5). (iii) Symptoms score improvements also were significant between preinjection and 2-week follow-up (P = 0.005) and preinjection and 8-week follow-up (P = 0.000; effect size, 1); there was also a significant deterioration between the 8-week and 24-week follow-up (P = 0.038), therefore, D24X Xthere D25X Xwas D26X Xno significantD27X XdifferenD28X Xce between pre-injection and 24-week follow-up (P = 0.483). Interestingly, the control group had significantly improved symptoms score between preinjection and 24-week follow-up (P = 0.040). (iv) S/R factor improvement was significant between preinjection and 8-week follow-up (P = 0.015; effect size, 0.8) and pre-injection and 24-week follow-up (P = 0.026; effect size, 0.9). The control group had significant improvement in S/R factors between pre-injection and 24-week follow-up (P = 0.048). (v) Pain improvement was significant between pre-injection and 2-week follow-up (P = 0.006), pre-injection and 8-week follow-up (P = 0.000; effect size, 1.4) and 2-week and 8-week follow-up (P = 0.035). Supplementary Figure 3 depicts the diagrams of VAS, ROM and the KOOS questionnaire evaluations.

MRA factors Of the total 28 points of cartilage thickness measurement in each patient, the cartilage thickness in the following items was significantly increased between pre-treatment and 24-week follow-up in the MSC group: superior medial patella maximum (SMPMax; P = 0.013; effect size, 0.2), middle medial patella maximum (MMP-Max; P = 0.025; effect size, 0.3) and tibial compartment, lateral minimum (TCL-Min; P = 0.011; effect size, 0.2). However, there were no significant changes during this interval in the control group (Figure 2). The distribution of synovial hypertrophy, spur, erosion or defects of ACL, medial and lateral meniscus among the two groups before intervention (yes/no) and the distribution of the defect behavior (stable/progress/regress) after intervention were evaluated using the chi-square test. In contrast to the pre-treatment comparison, there were significant differences between these para meters among the two groups (P = 0.000) after treatment. Table 3 lists the distribution percentages of the compared parameters except chondral thickness at the 24-week follow-up MRA in the two groups.

ARTICLE IN PRESS Allogenic stem cell therapy in knee osteoarthritis

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Table III. Comparison of parameters in percentage at the 24-week MRA. Erosion

Stable Progress Regress

Spur

Synovial hypertrophy

ACL lesions

Meniscus lesions

MSC

Placebo

MSC

Placebo

MSC

Placebo

MSC

Placebo

MSC

Placebo

40 50 10

60 30 10

90 10 0

100 0 0

70 30 0

70 20 10

60 40 0

100 0 0

100 0 0

100 0 0

Distribution percentage of qualitative parameters defined as stable, progress or regress at the 24-week follow-up MRA evaluation of the two groups.

Discussion To date, most of the published clinical trials have reported that autologous MSC therapy is a safe and promisingly effective method for treating OA [24]. In this clinical trial, we evaluated the clinical and para-clinical safety and efficacy of allogenic PLMSC intra-articular injection in 10 patients with mild to advanced knee OA as compared with 10 control patients. The allogenic PLMSC significantly improved the clinical measures of pain, symptoms, ADL, QOL, S/R factors and knee ROM. Interestingly, pain reduction, ADL and symptom improvement occurred as soon as 2 weeks after treatment and were sustained until 8 weeks post-treatment. Similar to our findings, almost all studies in which MSCs were applied to treat knee OA reported variable clinical improvements [14,24]. Our KOOS questionnaire evaluation showed that improvement ceased after 8 weeks. Similarly, Davatchi et al. and Emadedin et al. reported gradual decrease in functional improvements after 24 weeks [33,34]. This finding may enforce the idea of multiple injections for more sustained improvements. Here, we could not observe significant pain improvement evaluated using VAS score. This inconsistency with the KOOS pain score may have arisen from the greater possibility of the VAS score to be biased based on the patient-physician relationship, especially in long-standing follow-ups, rendering it a less precise score for pain measurement in comparison with the nine questions of the KOOS questionnaire about pain, which the patient answers independently instead of being interviewed by the physician. The literature also shows that the validity of VAS score is questionable in chronic musculoskeletal pain [35]. The improvement in joint ROM was the most significant clinical improvement in the intervention group, observed subjectively and objectively as early as 2 weeks. This primary pain reduction and ROM improvement can be related to the immunomodulatory and anti-inflammatory action rather than trophic action of MSCs [36 39].

Notably, we observed clinical improvement of pain, KOOS and ROM in the control group, despite lack of statically significant improvement. In some reported articles the placebo effect of normal saline injection yielded statistically and clinically significant improvement in patient-reported outcomes up to 24 weeks in patients with knee OA [40,41]. We do not have any definite scientific explanation for this observation. We used MRA evaluation to increase the sensitivity and specificity of chondral thickness quantification [42,43]. Chondral thickness was increased in approximately 10% of total knee joint cartilage areas measured among the patients who had received MSCs. SMP-Max, MMP-Max and TCL-Min were the areas in which chondral thickness increased significantly. Although there were radiologically unremarkable changes, this amount of radiological improvement was related to the significant clinical satisfaction mentioned earlier. Additionally, radiological improvement may be better observed at follow-up time points closer to injection time (e.g., 12 weeks instead of 24 weeks) to check for maximal chondral regenerative response. Most patients, irrespective of intervention group, were stable for synovial hypertrophy, spur, erosion and ACL, medial and lateral meniscus injuries. Regeneration of ACL tissue and meniscal lesions in response to the MSC injection still needs further investigations considering available studies with controversial reports [28,44,45]. Most studies showed that ACL or meniscus regeneration better takes place in scaffold-based MSC delivery and by differentiated cartilage cells compared with MSCs [46,47]. In this clinical trial we only observed self-limited local pain and swelling in a few patients of our case group as reported by some other trials [27,48]. There was no evidence of ectopic tissue, tumor formation, pulmonary embolism or liver or renal impairment at 24 weeks follow-up. Hematologic, biochemical and serological parameters all were the same as before injection. Other studies that used placental or allogenic MSCs with long follow-ups also support their safe usage [22,27,48 51].

ARTICLE IN PRESS 8 S. KHALIFEH SOLTANI et al. Low expression of major histocompatibility complex (MHC) proteins and T-cell co-stimulatory molecules in MSCs allow systemic and local application of these cells without life-threatening concerns of adverse immunogenicity [52]. Murphy et al. reported lower chondrogenic and adipogenic BM MSC potency derived from patients with OA than from healthy people [53]. Deterioration of stem cell renewal ability and differentiation potency by the aging process have also been shown [54,55]. These observations justify use of potent allogenic PLMSCs for OA treatment. We used placental MSCs, which provide an abundant supply of MSCs, circumventing the need to harvest stem cells by invasive procedures such as liposuction or bone marrow aspiration. Our study proves the safety of local infusion using unmatched allogeneic placental MSCs. In conclusion, intra-articular injection of allogenic PLMSCs is safe and can open optimistic views for managing OA. Pain improvement and increased knee ROM were the most significant clinical findings. Because OA is an ongoing process of joint destruction, the idea that multiple injections may improve and prolong the efficacy of MSC therapyD9X2 X is encouraging. Due to sample size limitation, we could not assess the effect of OA severity on treatment efficacy. Further largescale clinical trials will shed valuable insight into the curative properties and long-term sustenance of placental stem cells in patients with OA.

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Acknowledgments This study was supported by grant number 943798 of the National Institute D30X XFor Medical Research Development (NIMAD) granted to M. Vasei. The authors thank the Babak Radiology Center team for their cooperation in performing the MRAs. Disclosure of interests: There is no relevant financial or nonfinancial conflict of interests in this article. References [1] Mutangadura GB. World Health Report 2002: Reducing Risks, Promoting Healthy Life: World Health Organization, Geneva, 2002, pp 1-250, Agricultural Economics. 2004;30 (2):170-2. [2] Moskowitz RW. The burden of osteoarthritis: clinical and quality-of-life issues. Am J Manag Care 2009;15(8 Suppl): S223–9. [3] Fransen M, Bridgett L, March L, Hoy D, Penserga E, Brooks P. The epidemiology of osteoarthritis in Asia. Int J Rheum Dis 2011;14(2):113–21. [4] Sandoughi M, Zakeri Z, Tehrani Banihashemi A, Davatchi F, Narouie B, Shikhzadeh A, et al. Prevalence of musculoskeletal disorders in southeastern Iran: a WHO ILAR

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Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jcyt.2018.11.003.