Haemophilia (2015), 21, 693–701
DOI: 10.1111/hae.12659
ORIGINAL ARTICLE Laboratory science
Experimental study on the role of intra-articular injection of MSCs on cartilage regeneration in haemophilia R . R A V A N B O D , * G . T O R K A M A N , * M . M O P H I D † and F . M O H A M M A D A L I ‡ *Department of Physical Therapy, Biomechanical Research Laboratory, Tarbiat Modares University; †Department of Histology, Baquiyatallah University of Medical Sciences; and ‡Department of Hematology, Tarbiat Modares University, Tehran, Iran
Summary. Mesenchymal stem cells (MSCs) therapy is a field in progress in cartilage repair strategies. We tried to investigate the functional properties of the joint and cartilage in experimental haemarthrosis (EH) after MSCs intra-articular (IA) injection. One millilitre of fresh autologous blood was injected twice a week for three consecutive weeks in three groups including control haemophilia 10 days (n = 8), control haemophilia 38 days (n = 8) and MSCs (n = 8) group. In later, 10 days after the end of IA blood injections, MSCs IA injection was performed. Eight animals received no treatment as the normal control group. Thirty-eight days after the end of IA blood injections, animals were sacrificed. Joint friction and stressrelaxation tests were done, inflammatory cytokines of synovial membrane and scanning electron microscopy of the cartilage assessed. Joint friction decreased in MSCs in comparison to other groups and was
significant with normal control group, (P = 0.011). The mechanical properties of cartilage showed no significant differences between groups. Tumour necrosis factor alpha and interleukin 1 beta decreased and IL-4 very slightly increased in MSCs in comparison to the time-matched control group. Scanning electron microscopy enabled acquisition of good structural properties of the surface and layers of the cartilage after MSCs injection. The hole induced in the medial plateau of the tibia bones, after inducing haemarthrosis, were covered with cartilage-like structure. The results showed that MSCs IA injection has some beneficial effects on cartilage structure and function in haemarthrosis model and is promising in patients with haemophilia.
Introduction
destroy the joints in later life. The term of sports or trauma-induced arthropathy is common as replacement therapy of clotting factors makes PWH live a more normal life [4,5]. Therefore, the need of invasive procedures like joint arthroplasty is still a predicament both in developing countries with low factor consumption per capita and also in developed countries. There are surgical methods to restore synovial joint function in osteoarthritis (OA). Arthroscopic debridement, microfracture, mosaicplasty, autologous chondrocyte transplantation (ACT) and tissue-engineered-based constructs have been implemented [6,7]. However, only limited reports are available for joint debridement in haemophilic arthropathy (HA) [8–11]. The drawbacks of cartilage methods are the paucity of the cell source [12], different mechanical environments of the cartilage harvested from a non-weightbearing area being transferred to a culture medium and finally implanted to a weight-bearing area [13], chondrocytes dedifferentiation and hypertrophy, lack of adhesion to the host tissue [14,15] and principally
Decades ago, lack of prophylaxis made patients with haemophilia (PWH) experience massive bleedings and compelled them to tackle intensive on-demand therapy until reminiscence of haemarthroses. Nowadays, despite prophylactic treatment, chronic microhaemorrhages into the joint or subchondral bone, the influence of different gene defect on haemarthrosis phenotype, disturbed cartilage loading and other unknown aetiologies could induce silent synovitis and cartilage degeneration [1–3]. Not a rare occurrence of very mild synovitis in the shed of prophylaxis regimens, particularly low-dose prophylaxis, could silently Correspondence: Giti Torkaman, Faculty of Medical Sciences, Tarbiat Modares University, Across Chamran and Jalal-al-Ahmad Highways, Tehran 14115-331, Iran. Tel: +98 21 82884509; fax: +98 21 88013030; e-mail: [email protected] Accepted after revision 21 January 2015 © 2015 John Wiley & Sons Ltd
Keywords: articular cartilage, biomechanics, friction, haemarthrosis, knee joint, mesenchymal stem cell
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large cartilage lesions either in OA or HA. The global objective of cartilage repair is providing proper amount of a more functional tissue with minimally invasive fashion, less costly and well suited to treat large cartilage lesions [16]. Instead of chondrocytes with the abovementioned problems, MSCs with essential characteristics of both self renewal and multilineage differentiation potential have led to be a great candidate in cartilage repair strategies [17,18]. It was reported that 20–50% of the total population of bone marrow (BM)-derived MSCs are tri-potent with an important subpopulation of chondroprogenitors [19]. The ectopically implantation of ex vivo transduced F-IX or F-VIII genes either with adeno-associated virus or MSCs transduced by simian immunodeficiency virus vectors have performed after a single joint bleed as a novel therapeutic approach to prevent haemophilic synovitis [20,21]. Moreover, feasibility of autologous BM-derived MSCs for haemophilic arthropathy was investigated by morphology and flow cytometric analysis and showed BM-derived MSCs of three PWH were capable of differentiating into chondrocytes, similarly to those in a healthy adult [22]. As previously showed, in-situ biomechanical and ex vivo biotribological parameters could throw light upon some aspects of structure–function relationships of the whole synovial joint [23]. Moreover, proinflammatory cytokines, particularly interleukin 1 beta (IL-1b) and tumour necrosis factor alpha (TNF-a) in synovium and synovial fluid contribute to cartilage damage [24–26] and anti-inflammatory cytokines including IL-4 and IL-10 could ameliorate this procedure [27]. On the other hand, It is well known that MSCs also possess immunomodulatory effect [28]. Therefore, this study mainly focused on investigating the effects of MSCs IA injection on articular cartilage restoration of function and synovial membrane inflammation after multiple but not only one experimental haemarthrosis (EH).
Methods and materials A total of 32 male albino rabbits (Razi Vaccine and Serum Research Institute Karaj, Tehran, Iran) were selected, eight in each control normal (Con), control haemophilia 10 days after the end of IA blood injections (ConHem10), control haemophilia 38 days after
the end of IA blood injections (ConHem38) and MSCs IA injection groups, (Table 1). The animals were housed separately under strict hygienic condition. Multiple injections into the animals heart, joint, medullary canal, muscle and skin (for antibiotic and anaesthetic therapies) made this procedure very invasive and unfortunately we lost many animals and ethically were not able to continue the increase in animals number for each group. The study was approved by the ethical commission of Tarbiat Modares University.
Experimental design Knee joint perimeter was measured prior to blood injections and prior to the animals’ death. After anaesthesia using, 1 mL of fresh autologous blood was aspirated form the animals’ heart and injected into the animals left knee joint using previously described method, two times per week for three consecutive weeks [23]. In MSCs group, 1.5–2 mL of BM was obtained from the animal’s tibia bone of the right lower limb about 3 weeks prior to MSCs IA injection. First, a 21 gauge needle with a slight twisting motion advanced into the tibial medullary canal above and medial to the insertion site of patellar tendon in the animal’s knee joint flexion position. Second, this needle was removed as it was blocked by spongiform bone and replaced with another 21 gauge syringe which was coated with heparin 5000 IU mL 1 (Heparin Sodium 5000 Units, Rotexmedica, GMBH, Germany). and the BM aspirated. All the procedure was performed under aseptic condition. Each animal received a single dose of penicillin G procaine 800 000 IU (60 000 IU kg 1) and gentamicin 80 mg (10 mg kg 1) after the aspiration of the BM. After cessation of IA blood injections, 10 days were considered to be as a wash-out period for blood and resolving the acute phase of haemarthrosis. This interval was organized to be concomitant with preparation of MSCs. Therefore, the ConHem38 is the timematched control of MSCs group, (Table 1). On the day of MSCs IA injection, that is to say, 10 days after the end of the total six blood injections, 1 mL of cultured MSCs containing 6–10 9 105 cells with sterile phosphate-buffered saline (PBS) was prepared and injected into the left knee joint of the rabbits. At the end of the procedure, the animals were sacrificed with Chloroform inhalation and anaesthetic
Table 1. Groups of the study. Group
Con
ConHem10
ConHem38
MSCs
Details
Received no treatment, (n = 8)
Sacrificed 10 days after cessation of IA blood injections, to study the extend of cartilage damage, (n = 8)
Sacrificed 38 days after cessation of IA blood injections, to study probable self repair and as the control for MSCs group, (n = 8)
Received MSCs IA injection, 10 days after cessation of IA blood injections. Sacrificed 38 days after cessation of IA blood injections, means 28 days after MSCs IA injection, (n = 8)
MSCs, mesenchymal stem cells; IA, intra-articular.
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drugs overdose. The knee joint friction test by the pendulum friction tester system was performed. As previously described, friction could be assessed by five different methods including Stanton equation, exponential and linear curve fitting damping slope and average logarithmic and linear decrements [23]. Then, the joint was opened and the removed synovial membrane stored at 80°C for further cytokines assessments. The biomechanical (stress-relaxation) test of the medial plateau of the tibia was done afterwards (Zwick Universal Testing Machine Zwick/Roell GmbH & Co., Ulm, Germany) as previously described method. The stress-relaxation test variables were cartilage maximum force, maximum stiffness, equilibrium force and equilibrium stiffness. Cartilage thickness was measured on the medial plateau of the tibia. [23].
Mesenchymal stem cells expansion BM aspirate was mixed with PBS and centrifuged for 5 min at 1000Xg. The supernatant was removed and pellets transferred to 75 cm2 cell culture flasks with 15 mL Dulbecco’s modified eagle’s medium (DMEM) containing 10% foetal bovine serum, 100 U mL 1 penicillin G and 100 mg streptomycin and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The culture medium was changed after 3 days. Then, the attached cells were fed with fresh culture medium (DMEM containing 10% foetal bovine serum). Thereafter, the culture medium was replaced every 2–3 days. After reaching 80% of confluence, the cells were sub-cultured to next passage by using 0.05% trypsin–EDTA for up to passage 2. Morphologic analysis was performed daily with inverted phase contrast microscopy. Mesenchymal stem cells formed a monolayer of adherent cells and looked like long spindle-shaped fibroblastic cells in culture medium. For safety assessment, bacteriological tests were performed on the samples after each passage, and before injection. The viability of cells was assessed by counting viable cells with Trypan blue in a Neubauer chamber.
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placed in PBS. PBS was mixed with problock protease inhibitor cocktail (ProBlockTM 50; EDTA Free; Gold Biotechnology, Inc., Saint Louis, MO, USA) according to the manufacturer’s instruction and homogenized (Heidolph Diax 900; Sigma-Aldrich, St Louis, Missouri, USA). The final homogenate was centrifuged at 4000 g for 15 min and the supernatant fluid removed and stored at 80°C for ELISA tests of IL-1b, TNF-a and IL-4 using commercially available kits (Casaba, Hubei, P.R.China). Data are expressed in pg mL 1.
Scanning electron microscopy Tibia bone was fixed with 10% formaldehyde and decalcified using formic acid. The medial plateau of the tibia submitted to increasing ethanol concentrations. The plateau embedded in paraffin and small cartilage fragments (0.3 9 0.3 cm) prepared and the remaining plateau sectioned to an average thickness of 5 micrometers. Both of the prepared and sectioned tissues metalized with palladium gold and observed under scanning electron microscope (Philips XL 30, Hillsboro, OR, USA).
Statistical analysis One-Sample Kolmogorov–Smirnov test was used to determine the normal distribution. All the variables had normal distribution, (P > 0.05). Paired t-test was used for joint perimeter measurements. One way ANOVA and the Tukey post hoc testing were used to detect statistical differences in the friction, biomechanical and inflammatory parameter between groups. Differences were considered to be statistically significant when P ≤ 0.05. The statistical power for Stanton and maximum force variables were between 95% and 97%.
Results In MSCs group, the joint perimeter, although not significant, was the least among other experimental groups, (Table 1). The right knee joint perimeter also increased in experimental groups (Table 2).
Enzyme-linked immunosorbent assay (ELISA) To investigate whether MSCs had an effect on synovial membrane inflammatory condition after experimental haemarthrosis, the frozen synovial tissue samples were weighed and 5 mg of each sample
Friction results The mean of pendulum cycle number to reach equilibrium was slightly but not significantly increased in MSCsLt joints (Fig. 1). The coefficient of friction
Table 2. Knee joint perimeter (mm).
Left Right
Con
ConHem10
ConHem38
Mesenchymal stem cells
9.69 0.65 9.71 0.57
10.98 1.49 9.90 0.62
11.21 0.77 9.92 0.53
10.57 1.15 10.11 0.76
Values in the table are means SEM.
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40
Lt
35
Rt Lt
0.2
30 25
Stanton
Cycle number
*
0.25
20 15 10
Rt
0.15 0.1 0.05
5 0
0 Con
ConHem10 ConHem38
MSC
*
ConHem10 ConHem38
0.02 0.015
Lt Rt
0.01 0.005 0
MSC
*
0.025 Avg linear decrement
0.025
Linear curve fiƫng
Con
0.02 Lt
0.015
Rt
0.01 0.005 0
Con
ConHem10 ConHem38
MSC
Con
ConHem10 ConHem38
MSC
Avg log decrement
0.035 0.03
Lt
0.025
Rt
0.02 0.015
Fig. 1. Friction estimation with different methods including pendulum cycle number to reach equilibrium, Stanton equation, linear curve fitting damping slope and average linear and logarithmic decrement. *P < 0.05.
0.01 0.005 0 Con
ConHem10 ConHem38
MSC
calculated by Stanton equation in comparison to control group was significantly lower in MSCsLt joints (P = 0.011). In Linear curve fitting damping slope and average linear decrement methods, friction also decreased significantly in MSCsLt group, (Fig. 1).
Tensiometer results All the results of the stress-relaxation data are presented in Table 2. No statistical significance was found in all the parameters of the Lt knees as the experimental joints, (Table 3). Cartilage thickness decreased in ConHemLt10 group (0.12 0.01 mm) and continued to decrease in ConHemLt38 (0.11 0.01 mm) but increased in MSCsLt group (0.19 0.02 mm) in comparison to normal control Lt knees (0.38 0.15 mm), (Fig. 2).
Scanning electron microscopy Cartilage surface architecture and integration could be seen in Fig. 4 and cartilage cells and depth in Fig. 5. Surface irregularities are depicted in ConHem10 and are remarkable in ConHem38. In MSCs group in comparison to its matched control, ConHem38, the surface irregularities resolved remarkably. In ConHem10 less active chondrocytes and apoptosis could be seen. Chondrocytes are irregularly arranged and the uppermost superficial layer is absorbed. In ConHem38, note the pronounced chondrocyte apoptosis and reduction in the cell number. The uppermost superficial layer is visible although with deep fissures. Viable chondrocytes in high quantity and normal distribution are seen in MSCs group, also note the intact uppermost superficial layer, Fig. 5.
Discussion Biochemical analysis Figure 3 shows the proinflammatory cytokines levels of TNF-a and IL-1b and anti-inflammatory cytokine IL-4 in the supernatant fluid of the synovial tissues. There was no statistically significant difference between the groups. Proinflammatory cytokines showed slightly lower activity in MSCs group in comparison to ConHem38. Haemophilia (2015), 21, 693--701
Previous studies clearly demonstrated that deterioration happens in different parts of synovial joint in the presence of blood [29–37]. Treatment of haemophilic severe arthropathy with advanced therapies has been recommended and is an area of particular interest [6,38]. In the present study, we tried to examine the effects of MSCs treatment in a condition more similar to © 2015 John Wiley & Sons Ltd
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Table 3. Tensiometer parameters in different groups. Max force (MPa) Control Lt 0.302 Rt 0.104 ConHem10 Lt 0.1167 Rt 0.1402 ConHem38 Lt 0.1008 Rt 0.143 Mesenchymal stem cells Lt 0.0965 Rt 0.0900
Stiffness (MPa)
Equilibrium force (MPa)
Equilibrium stiffness (MPa)
0.077 0.019
4.851 1.375 1.513 0.339
0.087 0.0173 0.056 0.0136
13.846 7.765 5.976 6.675
0.0176 0.0352
1.277 0.214 1.525 0.409
0.0954 0.032 0.0499 0.015
4.746 0.649 4.478 0.595
0.0201 0.033
0.974 0.266 1.446 0.337
0.0658 0.0137 0.0666 0.008
4.530 1.124 4.684 0.595
0.0136 0.0141
0.948 0.145 0.889 0.160
0.0693 0.0147 0.492 0.0086
4.239 0.485 3.738 0.279
Values are means SEM.
*
0.5 0.45
Cartilage thickness (mm)
0.4 0.35 0.3
Lt Rt
0.25 0.2 0.15 0.1 0.05 0
Con
ConHem10
ConHem38
MSC
Fig. 2. Cartilage thickness. *P < 0.05.
TNF-α
IL-1β
IL-4
60
2.5
50
2
45 40
1.5
35 30
1
25 20
IL-4 (pg mL–1)
TNF-α, IL-1β (pg mL–1)
55
0.5
15 10
Con
ConHem10
ConHem38
MSC
0
Fig. 3. Pro and anti-inflammatory cytokines.
what happens in haemophilic joints rather than a single bleeding induced by joint capsular puncture in which the amount of bleeding seems not to be always © 2015 John Wiley & Sons Ltd
determined accurately and the joint and cartilage do not have a reasonable exposure time to blood. Kashiwakura et al. perfectly investigated the effects of the transduced MSCs containing the F-VIII gene by Valentino et al. quantitative grading system of synovitis with only one score relating to cartilage erosion [20,38]. We tried to examine the possible effects of MSCs intrinsic regenerative capacity and not their gene transduced forms. Although the chondrogenic differentiation and matrix synthesis stimulation of MSCs could be influenced by transforming growth factor b (TGF-b) family [39–41], Chang et al. concluded that using undifferentiated MSCs might be a better approach for in vivo osteochondral defects. This evidence confirmed by capturing TGF-b with an antibody did not result in significant changes in cartilage-related gene expression of BM MSCs. Accordingly, for the first steps of MSCs usage in EH, we determined not to use a differentiation factor [42,43]. Friction, Lubrication and stiffness cannot be measured in vivo and can be estimated with subjective tests in clinical practices. Most of the outcome measures including WOMAC questionnaire are subjective [44– 46]. Pendulum friction tester provides a quantitative ex vivo measurement of joint friction and lubrication. The significant decrease in COF in MSCs group of the present study was consistent with the significant COF decrease in equine synovial fluid soaked, MSCs alginate construct by 10% after 4 weeks of MSCs culture and by 20% after 6 weeks of MSCS culture [47]. Moreover, the decrease in COF might be as a consequence of slight decrease in IL-1b and TNF-a in MSCs group [48]. The objectives of cartilage repair are support and load distribution as mechanical properties and low friction provided by proper lubrication and normal cartilage structure. Therefore, the significant decrease in COF after MSCs IA injection is a good finding for HA as one of the chief complaints for accepting joint arthroplasty in PWH is stiffness and the resultant starting pain. Despite reasonable increase in cartilage thickness in MSCs group none of the biomechanical parameters Haemophilia (2015), 21, 693--701
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ConHem 10
MSCs
ConHem 38n
Control
Fig. 4. Electromicrograph of the cartilage surface architecture in different groups. Bars: 100 lm.
had reasonable sign of healing. The biomechanical results are in accordance with Ando et al. study demonstrated compromised mechanical properties with normal friction in scaffold free tissue-engineered construct derived from synovial MSCs [49]. It is known that the adult stem cells secrete hyaluronic acid, lubricin and keratan sulphate which could reduce joint friction [50,51]. The interesting macroscopic finding was a hole in almost all medial plateau of the tibia bones (Fig. 6). This hole was not surgically provoked and was due to the effect of blood and postulated that probably induced as the line of gravity predominantly passes through medial compartment of the knee joint [52,53]. Therefore, the indentation tests of control groups (including ConHem10 and ConHem38) first performed on the hole but as the hole contained no cartilage, the Haemophilia (2015), 21, 693--701
probe faced the subchondral bone and any results from the cartilage structure could not be obtained. Consequently, the area around the hole considered for biomechanical evaluation of the control groups. However, by the time of joint opening in MSCs group, a newly formed cartilage-like structure was detected on the hole area and biomechanical properties of this newly formed cartilage-like structure was measured. Therefore, the biomechanical parameters results of MSCs group of this study, although seems not to be beneficial (with the exemption of cartilage thickness), definitely is superior to subchondral bone. Proinflammatory cytokines like TNF-a and IL-1b have prominent role in blood-induced joint damage [54]. TNF-a showed a reasonable decrease in the MSCs in comparison to the other groups and IL-1b was higher than Con, but slightly lower than Con© 2015 John Wiley & Sons Ltd
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ConHem 10
ConHem 38
MSCs
Control
699
Fig. 5. Electromicrograph of the hyaline cartilage of different groups. Bars: 100 lm.
Fig. 6. Hole in the medial plateau of tibia bone of ConHem38 group (left), Repaired hole in mesenchymal stem cells group (right).
Hem38. The role of anti-inflammatory cytokines as specific therapies has gained attention in preventing BIJD in parallel with clotting factor replacement [27,55,56]. Il-4 slightly increased in MSCs in comparison to its control group and could reveal that MSCs are capable of gently touching the anti-inflammatory condition of the EH damaged joint. Greish et al., found that TNF-a and Il-1b decreased significantly in © 2015 John Wiley & Sons Ltd
MSCs group in comparison to control in complete Freunds adjuvant induced arthritis of rats hind paw and IL-10 increased significantly. However, the cytokine assays performed in the rat serum and the arthritis model was not confined to a joint space like the present study [57]. The SEM results showed MSCs could regenerate chondrocytes despite the fact that the force and stiffness of the newly formed cartilage were low. One explanation is that the chondrocytes did not have plenty of time to produce more cartilage matrix to be able to withstand loading. There might have been a possibility for further improvement of the mechanical properties of the cartilage with a longer follow up. The scanning electron micrograph of ConHem38 depicts the breakdown of cartilage structure and confirms the long-term potential of blood in cartilage damage [29,30]. This amount of arthropathy in an animal model with normal coagulation conveys the Haemophilia (2015), 21, 693--701
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importance of prophylaxis and the need of new kinds of treatment like MSCs therapy in PWH. Safety is an important prerequisite for MSCs therapy, particularly in orthopaedic field which is not life threatening. The BM-derived MSCs were checked for tumour development and infections in 41 patients received 45 transplantation and neither tumour nor infections were observed for a mean 75 (5–137) months of following up [58]. Based on Peeters et al. review on the safety of IA cell-therapy with cultureexpanded stem cells, this application reported to be safe [59]. Moreover, age-related biological characterization of MSCs in a human articular cartilage study showed spontaneous osteogenic differentiation besides lower chondrogenic differentiation only in elderly patients. As HA predominantly happens in younger ages, It would be expected to find more reasonable results through MSCs IA injection [60]. In conclusion, this study shows that haemarthrosis if left untreated could lead to severe cartilage damage. MSCs IA injection after EH could reduce
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Acknowledgements The authors would like to thank Prof Abron for his support in MSCs culture. This study was supported by a grant from presidency of the Islamic Republic of Iran, vice-presidency for science and technology. The study sponsor had no involvement in the research procedure or writing the manuscript or in the decision to submit the manuscript for publication.
Disclosures The authors stated that they had no interests which might be perceived as posing a conflict or bias.
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sion of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells. Biochem J 2001; 354(Pt 1): 17–24. Tew SR, Murdoch AD, Rauchenberg RP, Hardingham TE. Cellular methods in cartilage research: primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells. Methods 2008; 45: 2–9. Chang CH, Kuo TF, Lin FH et al. Tissue engineering-based cartilage repair with mesenchymal stem cells in a porcine model. J Orthop Res 2011; 29: 1874–80. de Vries-van Melle ML, Narcisi R, Kops N et al. Chondrogenesis of mesenchymal stem cells in an osteochondral environment is mediated by the subchondral bone. Tissue Eng Part A 2014; 20: 23–33. Zelada F, de Almeida AM, Pailo AF, Bolliger R, Okazaki E, de Rezende MU. Viscosupplementation in patients with hemophilic arthropathy. Acta Ortop Bras 2013; 21: 12–7. Zingg PO, Fucentese SF, Lutz W, Brand B, Mamisch N, Koch PP. Haemophilic knee arthropathy: long-term outcome after total knee replacement. Knee Surg Sports Traumatol Arthrosc 2012; 20: 2465–70. Viliani T, Zambelan G, Pandolfi C et al. In-patient rehabilitation in haemophilic subjects with total knee arthroplasty. Haemophilia 2011; 17: e999–1004. Gleghorn JP, Jones AR, Flannery CR, Bonassar LJ. Boundary mode frictional properties of engineered cartilaginous tissues. Eur Cell Mater 2007; 14: 20–8. Gleghorn JP, Jones AR, Flannery CR, Bonassar LJ. Alteration of articular cartilage frictional properties by transforming growth factor beta, interleukin-1beta, and oncostatin M. Arthritis Rheum 2009; 60: 440–9. Ando W, Fujie H, Moriguchi Y et al. Detection of abnormalities in the superficial zone of cartilage repaired using a tissue engineered construct derived from synovial stem cells. Eur Cell Mater 2012; 24: 292– 307. Meng QG, Long X. A hypothetical biological synovial fluid for treatment of temporomandibular joint disease. Med Hypotheses 2008; 70: 835–7.
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51 Greene GW, Banquy X, Lee DW, Lowrey DD, Yu J, Israelachvili JN. Adaptive mechanically controlled lubrication mechanism found in articular joints. Proc Natl Acad Sci USA 2011; 108: 5255–9. 52 Staubli AE, Jacob HA. Evolution of openwedge high-tibial osteotomy: experience with a special angular stable device for internal fixation without interposition material. Int Orthop 2010; 34: 167–72. 53 Hsu RW, Himeno S, Coventry MB, Chao EY. Normal axial alignment of the lower extremity and load-bearing distribution at the knee. Clin Orthop Relat Res 1990; 255: 215–27. 54 Forsyth AL, Rivard GE, Valentino LA et al. Consequences of intra-articular bleeding in haemophilia: science to clinical practice and beyond. Haemophilia 2012; 18(Suppl. 4): 112–9. 55 Jansen NW, Roosendaal G, Hooiveld MJ et al. Interleukin-10 protects against bloodinduced joint damage. Br J Haematol 2008; 142: 953–61. 56 Narkbunnam N, Sun J, Hu G et al. IL-6 receptor antagonist as adjunctive therapy with clotting factor replacement to protect against bleeding-induced arthropathy in hemophilia. J Thromb Haemost 2013; 11: 881–93. 57 Greish S, Abogresha N, Abdel-Hady Z, Zakaria E, Ghaly M, Hefny M. Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model. World J Stem Cells 2012; 4: 101–9. 58 Wakitani S, Okabe T, Horibe S et al. Safety of autologous bone marrow-derived mesenchymal stem cell transplantation for cartilage repair in 41 patients with 45 joints followed for up to 11 years and 5 months. J Tissue Eng Regen Med 2011; 5: 146–50. 59 Peeters CM, Leijs MJ, Reijman M, van Osch GJ, Bos PK. Safety of intra-articular cell-therapy with culture-expanded stem cells in humans: a systematic literature review. Osteoarthritis Cartilage 2013; 21: 1465–73. 60 Chang HX, Yang L, Li Z, Chen G, Dai G. Age-related biological characterization of mesenchymal progenitor cells in human articular cartilage. Orthopedics 2011; 34: e382–8.
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DOI: 10.1111/hae.12659
ORIGINAL ARTICLE Laboratory science
Experimental study on the role of intra-articular injection of MSCs on cartilage regeneration in haemophilia R . R A V A N B O D , * G . T O R K A M A N , * M . M O P H I D † and F . M O H A M M A D A L I ‡ *Department of Physical Therapy, Biomechanical Research Laboratory, Tarbiat Modares University; †Department of Histology, Baquiyatallah University of Medical Sciences; and ‡Department of Hematology, Tarbiat Modares University, Tehran, Iran
Summary. Mesenchymal stem cells (MSCs) therapy is a field in progress in cartilage repair strategies. We tried to investigate the functional properties of the joint and cartilage in experimental haemarthrosis (EH) after MSCs intra-articular (IA) injection. One millilitre of fresh autologous blood was injected twice a week for three consecutive weeks in three groups including control haemophilia 10 days (n = 8), control haemophilia 38 days (n = 8) and MSCs (n = 8) group. In later, 10 days after the end of IA blood injections, MSCs IA injection was performed. Eight animals received no treatment as the normal control group. Thirty-eight days after the end of IA blood injections, animals were sacrificed. Joint friction and stressrelaxation tests were done, inflammatory cytokines of synovial membrane and scanning electron microscopy of the cartilage assessed. Joint friction decreased in MSCs in comparison to other groups and was
significant with normal control group, (P = 0.011). The mechanical properties of cartilage showed no significant differences between groups. Tumour necrosis factor alpha and interleukin 1 beta decreased and IL-4 very slightly increased in MSCs in comparison to the time-matched control group. Scanning electron microscopy enabled acquisition of good structural properties of the surface and layers of the cartilage after MSCs injection. The hole induced in the medial plateau of the tibia bones, after inducing haemarthrosis, were covered with cartilage-like structure. The results showed that MSCs IA injection has some beneficial effects on cartilage structure and function in haemarthrosis model and is promising in patients with haemophilia.
Introduction
destroy the joints in later life. The term of sports or trauma-induced arthropathy is common as replacement therapy of clotting factors makes PWH live a more normal life [4,5]. Therefore, the need of invasive procedures like joint arthroplasty is still a predicament both in developing countries with low factor consumption per capita and also in developed countries. There are surgical methods to restore synovial joint function in osteoarthritis (OA). Arthroscopic debridement, microfracture, mosaicplasty, autologous chondrocyte transplantation (ACT) and tissue-engineered-based constructs have been implemented [6,7]. However, only limited reports are available for joint debridement in haemophilic arthropathy (HA) [8–11]. The drawbacks of cartilage methods are the paucity of the cell source [12], different mechanical environments of the cartilage harvested from a non-weightbearing area being transferred to a culture medium and finally implanted to a weight-bearing area [13], chondrocytes dedifferentiation and hypertrophy, lack of adhesion to the host tissue [14,15] and principally
Decades ago, lack of prophylaxis made patients with haemophilia (PWH) experience massive bleedings and compelled them to tackle intensive on-demand therapy until reminiscence of haemarthroses. Nowadays, despite prophylactic treatment, chronic microhaemorrhages into the joint or subchondral bone, the influence of different gene defect on haemarthrosis phenotype, disturbed cartilage loading and other unknown aetiologies could induce silent synovitis and cartilage degeneration [1–3]. Not a rare occurrence of very mild synovitis in the shed of prophylaxis regimens, particularly low-dose prophylaxis, could silently Correspondence: Giti Torkaman, Faculty of Medical Sciences, Tarbiat Modares University, Across Chamran and Jalal-al-Ahmad Highways, Tehran 14115-331, Iran. Tel: +98 21 82884509; fax: +98 21 88013030; e-mail: [email protected] Accepted after revision 21 January 2015 © 2015 John Wiley & Sons Ltd
Keywords: articular cartilage, biomechanics, friction, haemarthrosis, knee joint, mesenchymal stem cell
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large cartilage lesions either in OA or HA. The global objective of cartilage repair is providing proper amount of a more functional tissue with minimally invasive fashion, less costly and well suited to treat large cartilage lesions [16]. Instead of chondrocytes with the abovementioned problems, MSCs with essential characteristics of both self renewal and multilineage differentiation potential have led to be a great candidate in cartilage repair strategies [17,18]. It was reported that 20–50% of the total population of bone marrow (BM)-derived MSCs are tri-potent with an important subpopulation of chondroprogenitors [19]. The ectopically implantation of ex vivo transduced F-IX or F-VIII genes either with adeno-associated virus or MSCs transduced by simian immunodeficiency virus vectors have performed after a single joint bleed as a novel therapeutic approach to prevent haemophilic synovitis [20,21]. Moreover, feasibility of autologous BM-derived MSCs for haemophilic arthropathy was investigated by morphology and flow cytometric analysis and showed BM-derived MSCs of three PWH were capable of differentiating into chondrocytes, similarly to those in a healthy adult [22]. As previously showed, in-situ biomechanical and ex vivo biotribological parameters could throw light upon some aspects of structure–function relationships of the whole synovial joint [23]. Moreover, proinflammatory cytokines, particularly interleukin 1 beta (IL-1b) and tumour necrosis factor alpha (TNF-a) in synovium and synovial fluid contribute to cartilage damage [24–26] and anti-inflammatory cytokines including IL-4 and IL-10 could ameliorate this procedure [27]. On the other hand, It is well known that MSCs also possess immunomodulatory effect [28]. Therefore, this study mainly focused on investigating the effects of MSCs IA injection on articular cartilage restoration of function and synovial membrane inflammation after multiple but not only one experimental haemarthrosis (EH).
Methods and materials A total of 32 male albino rabbits (Razi Vaccine and Serum Research Institute Karaj, Tehran, Iran) were selected, eight in each control normal (Con), control haemophilia 10 days after the end of IA blood injections (ConHem10), control haemophilia 38 days after
the end of IA blood injections (ConHem38) and MSCs IA injection groups, (Table 1). The animals were housed separately under strict hygienic condition. Multiple injections into the animals heart, joint, medullary canal, muscle and skin (for antibiotic and anaesthetic therapies) made this procedure very invasive and unfortunately we lost many animals and ethically were not able to continue the increase in animals number for each group. The study was approved by the ethical commission of Tarbiat Modares University.
Experimental design Knee joint perimeter was measured prior to blood injections and prior to the animals’ death. After anaesthesia using, 1 mL of fresh autologous blood was aspirated form the animals’ heart and injected into the animals left knee joint using previously described method, two times per week for three consecutive weeks [23]. In MSCs group, 1.5–2 mL of BM was obtained from the animal’s tibia bone of the right lower limb about 3 weeks prior to MSCs IA injection. First, a 21 gauge needle with a slight twisting motion advanced into the tibial medullary canal above and medial to the insertion site of patellar tendon in the animal’s knee joint flexion position. Second, this needle was removed as it was blocked by spongiform bone and replaced with another 21 gauge syringe which was coated with heparin 5000 IU mL 1 (Heparin Sodium 5000 Units, Rotexmedica, GMBH, Germany). and the BM aspirated. All the procedure was performed under aseptic condition. Each animal received a single dose of penicillin G procaine 800 000 IU (60 000 IU kg 1) and gentamicin 80 mg (10 mg kg 1) after the aspiration of the BM. After cessation of IA blood injections, 10 days were considered to be as a wash-out period for blood and resolving the acute phase of haemarthrosis. This interval was organized to be concomitant with preparation of MSCs. Therefore, the ConHem38 is the timematched control of MSCs group, (Table 1). On the day of MSCs IA injection, that is to say, 10 days after the end of the total six blood injections, 1 mL of cultured MSCs containing 6–10 9 105 cells with sterile phosphate-buffered saline (PBS) was prepared and injected into the left knee joint of the rabbits. At the end of the procedure, the animals were sacrificed with Chloroform inhalation and anaesthetic
Table 1. Groups of the study. Group
Con
ConHem10
ConHem38
MSCs
Details
Received no treatment, (n = 8)
Sacrificed 10 days after cessation of IA blood injections, to study the extend of cartilage damage, (n = 8)
Sacrificed 38 days after cessation of IA blood injections, to study probable self repair and as the control for MSCs group, (n = 8)
Received MSCs IA injection, 10 days after cessation of IA blood injections. Sacrificed 38 days after cessation of IA blood injections, means 28 days after MSCs IA injection, (n = 8)
MSCs, mesenchymal stem cells; IA, intra-articular.
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drugs overdose. The knee joint friction test by the pendulum friction tester system was performed. As previously described, friction could be assessed by five different methods including Stanton equation, exponential and linear curve fitting damping slope and average logarithmic and linear decrements [23]. Then, the joint was opened and the removed synovial membrane stored at 80°C for further cytokines assessments. The biomechanical (stress-relaxation) test of the medial plateau of the tibia was done afterwards (Zwick Universal Testing Machine Zwick/Roell GmbH & Co., Ulm, Germany) as previously described method. The stress-relaxation test variables were cartilage maximum force, maximum stiffness, equilibrium force and equilibrium stiffness. Cartilage thickness was measured on the medial plateau of the tibia. [23].
Mesenchymal stem cells expansion BM aspirate was mixed with PBS and centrifuged for 5 min at 1000Xg. The supernatant was removed and pellets transferred to 75 cm2 cell culture flasks with 15 mL Dulbecco’s modified eagle’s medium (DMEM) containing 10% foetal bovine serum, 100 U mL 1 penicillin G and 100 mg streptomycin and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The culture medium was changed after 3 days. Then, the attached cells were fed with fresh culture medium (DMEM containing 10% foetal bovine serum). Thereafter, the culture medium was replaced every 2–3 days. After reaching 80% of confluence, the cells were sub-cultured to next passage by using 0.05% trypsin–EDTA for up to passage 2. Morphologic analysis was performed daily with inverted phase contrast microscopy. Mesenchymal stem cells formed a monolayer of adherent cells and looked like long spindle-shaped fibroblastic cells in culture medium. For safety assessment, bacteriological tests were performed on the samples after each passage, and before injection. The viability of cells was assessed by counting viable cells with Trypan blue in a Neubauer chamber.
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placed in PBS. PBS was mixed with problock protease inhibitor cocktail (ProBlockTM 50; EDTA Free; Gold Biotechnology, Inc., Saint Louis, MO, USA) according to the manufacturer’s instruction and homogenized (Heidolph Diax 900; Sigma-Aldrich, St Louis, Missouri, USA). The final homogenate was centrifuged at 4000 g for 15 min and the supernatant fluid removed and stored at 80°C for ELISA tests of IL-1b, TNF-a and IL-4 using commercially available kits (Casaba, Hubei, P.R.China). Data are expressed in pg mL 1.
Scanning electron microscopy Tibia bone was fixed with 10% formaldehyde and decalcified using formic acid. The medial plateau of the tibia submitted to increasing ethanol concentrations. The plateau embedded in paraffin and small cartilage fragments (0.3 9 0.3 cm) prepared and the remaining plateau sectioned to an average thickness of 5 micrometers. Both of the prepared and sectioned tissues metalized with palladium gold and observed under scanning electron microscope (Philips XL 30, Hillsboro, OR, USA).
Statistical analysis One-Sample Kolmogorov–Smirnov test was used to determine the normal distribution. All the variables had normal distribution, (P > 0.05). Paired t-test was used for joint perimeter measurements. One way ANOVA and the Tukey post hoc testing were used to detect statistical differences in the friction, biomechanical and inflammatory parameter between groups. Differences were considered to be statistically significant when P ≤ 0.05. The statistical power for Stanton and maximum force variables were between 95% and 97%.
Results In MSCs group, the joint perimeter, although not significant, was the least among other experimental groups, (Table 1). The right knee joint perimeter also increased in experimental groups (Table 2).
Enzyme-linked immunosorbent assay (ELISA) To investigate whether MSCs had an effect on synovial membrane inflammatory condition after experimental haemarthrosis, the frozen synovial tissue samples were weighed and 5 mg of each sample
Friction results The mean of pendulum cycle number to reach equilibrium was slightly but not significantly increased in MSCsLt joints (Fig. 1). The coefficient of friction
Table 2. Knee joint perimeter (mm).
Left Right
Con
ConHem10
ConHem38
Mesenchymal stem cells
9.69 0.65 9.71 0.57
10.98 1.49 9.90 0.62
11.21 0.77 9.92 0.53
10.57 1.15 10.11 0.76
Values in the table are means SEM.
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40
Lt
35
Rt Lt
0.2
30 25
Stanton
Cycle number
*
0.25
20 15 10
Rt
0.15 0.1 0.05
5 0
0 Con
ConHem10 ConHem38
MSC
*
ConHem10 ConHem38
0.02 0.015
Lt Rt
0.01 0.005 0
MSC
*
0.025 Avg linear decrement
0.025
Linear curve fiƫng
Con
0.02 Lt
0.015
Rt
0.01 0.005 0
Con
ConHem10 ConHem38
MSC
Con
ConHem10 ConHem38
MSC
Avg log decrement
0.035 0.03
Lt
0.025
Rt
0.02 0.015
Fig. 1. Friction estimation with different methods including pendulum cycle number to reach equilibrium, Stanton equation, linear curve fitting damping slope and average linear and logarithmic decrement. *P < 0.05.
0.01 0.005 0 Con
ConHem10 ConHem38
MSC
calculated by Stanton equation in comparison to control group was significantly lower in MSCsLt joints (P = 0.011). In Linear curve fitting damping slope and average linear decrement methods, friction also decreased significantly in MSCsLt group, (Fig. 1).
Tensiometer results All the results of the stress-relaxation data are presented in Table 2. No statistical significance was found in all the parameters of the Lt knees as the experimental joints, (Table 3). Cartilage thickness decreased in ConHemLt10 group (0.12 0.01 mm) and continued to decrease in ConHemLt38 (0.11 0.01 mm) but increased in MSCsLt group (0.19 0.02 mm) in comparison to normal control Lt knees (0.38 0.15 mm), (Fig. 2).
Scanning electron microscopy Cartilage surface architecture and integration could be seen in Fig. 4 and cartilage cells and depth in Fig. 5. Surface irregularities are depicted in ConHem10 and are remarkable in ConHem38. In MSCs group in comparison to its matched control, ConHem38, the surface irregularities resolved remarkably. In ConHem10 less active chondrocytes and apoptosis could be seen. Chondrocytes are irregularly arranged and the uppermost superficial layer is absorbed. In ConHem38, note the pronounced chondrocyte apoptosis and reduction in the cell number. The uppermost superficial layer is visible although with deep fissures. Viable chondrocytes in high quantity and normal distribution are seen in MSCs group, also note the intact uppermost superficial layer, Fig. 5.
Discussion Biochemical analysis Figure 3 shows the proinflammatory cytokines levels of TNF-a and IL-1b and anti-inflammatory cytokine IL-4 in the supernatant fluid of the synovial tissues. There was no statistically significant difference between the groups. Proinflammatory cytokines showed slightly lower activity in MSCs group in comparison to ConHem38. Haemophilia (2015), 21, 693--701
Previous studies clearly demonstrated that deterioration happens in different parts of synovial joint in the presence of blood [29–37]. Treatment of haemophilic severe arthropathy with advanced therapies has been recommended and is an area of particular interest [6,38]. In the present study, we tried to examine the effects of MSCs treatment in a condition more similar to © 2015 John Wiley & Sons Ltd
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Table 3. Tensiometer parameters in different groups. Max force (MPa) Control Lt 0.302 Rt 0.104 ConHem10 Lt 0.1167 Rt 0.1402 ConHem38 Lt 0.1008 Rt 0.143 Mesenchymal stem cells Lt 0.0965 Rt 0.0900
Stiffness (MPa)
Equilibrium force (MPa)
Equilibrium stiffness (MPa)
0.077 0.019
4.851 1.375 1.513 0.339
0.087 0.0173 0.056 0.0136
13.846 7.765 5.976 6.675
0.0176 0.0352
1.277 0.214 1.525 0.409
0.0954 0.032 0.0499 0.015
4.746 0.649 4.478 0.595
0.0201 0.033
0.974 0.266 1.446 0.337
0.0658 0.0137 0.0666 0.008
4.530 1.124 4.684 0.595
0.0136 0.0141
0.948 0.145 0.889 0.160
0.0693 0.0147 0.492 0.0086
4.239 0.485 3.738 0.279
Values are means SEM.
*
0.5 0.45
Cartilage thickness (mm)
0.4 0.35 0.3
Lt Rt
0.25 0.2 0.15 0.1 0.05 0
Con
ConHem10
ConHem38
MSC
Fig. 2. Cartilage thickness. *P < 0.05.
TNF-α
IL-1β
IL-4
60
2.5
50
2
45 40
1.5
35 30
1
25 20
IL-4 (pg mL–1)
TNF-α, IL-1β (pg mL–1)
55
0.5
15 10
Con
ConHem10
ConHem38
MSC
0
Fig. 3. Pro and anti-inflammatory cytokines.
what happens in haemophilic joints rather than a single bleeding induced by joint capsular puncture in which the amount of bleeding seems not to be always © 2015 John Wiley & Sons Ltd
determined accurately and the joint and cartilage do not have a reasonable exposure time to blood. Kashiwakura et al. perfectly investigated the effects of the transduced MSCs containing the F-VIII gene by Valentino et al. quantitative grading system of synovitis with only one score relating to cartilage erosion [20,38]. We tried to examine the possible effects of MSCs intrinsic regenerative capacity and not their gene transduced forms. Although the chondrogenic differentiation and matrix synthesis stimulation of MSCs could be influenced by transforming growth factor b (TGF-b) family [39–41], Chang et al. concluded that using undifferentiated MSCs might be a better approach for in vivo osteochondral defects. This evidence confirmed by capturing TGF-b with an antibody did not result in significant changes in cartilage-related gene expression of BM MSCs. Accordingly, for the first steps of MSCs usage in EH, we determined not to use a differentiation factor [42,43]. Friction, Lubrication and stiffness cannot be measured in vivo and can be estimated with subjective tests in clinical practices. Most of the outcome measures including WOMAC questionnaire are subjective [44– 46]. Pendulum friction tester provides a quantitative ex vivo measurement of joint friction and lubrication. The significant decrease in COF in MSCs group of the present study was consistent with the significant COF decrease in equine synovial fluid soaked, MSCs alginate construct by 10% after 4 weeks of MSCs culture and by 20% after 6 weeks of MSCS culture [47]. Moreover, the decrease in COF might be as a consequence of slight decrease in IL-1b and TNF-a in MSCs group [48]. The objectives of cartilage repair are support and load distribution as mechanical properties and low friction provided by proper lubrication and normal cartilage structure. Therefore, the significant decrease in COF after MSCs IA injection is a good finding for HA as one of the chief complaints for accepting joint arthroplasty in PWH is stiffness and the resultant starting pain. Despite reasonable increase in cartilage thickness in MSCs group none of the biomechanical parameters Haemophilia (2015), 21, 693--701
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ConHem 10
MSCs
ConHem 38n
Control
Fig. 4. Electromicrograph of the cartilage surface architecture in different groups. Bars: 100 lm.
had reasonable sign of healing. The biomechanical results are in accordance with Ando et al. study demonstrated compromised mechanical properties with normal friction in scaffold free tissue-engineered construct derived from synovial MSCs [49]. It is known that the adult stem cells secrete hyaluronic acid, lubricin and keratan sulphate which could reduce joint friction [50,51]. The interesting macroscopic finding was a hole in almost all medial plateau of the tibia bones (Fig. 6). This hole was not surgically provoked and was due to the effect of blood and postulated that probably induced as the line of gravity predominantly passes through medial compartment of the knee joint [52,53]. Therefore, the indentation tests of control groups (including ConHem10 and ConHem38) first performed on the hole but as the hole contained no cartilage, the Haemophilia (2015), 21, 693--701
probe faced the subchondral bone and any results from the cartilage structure could not be obtained. Consequently, the area around the hole considered for biomechanical evaluation of the control groups. However, by the time of joint opening in MSCs group, a newly formed cartilage-like structure was detected on the hole area and biomechanical properties of this newly formed cartilage-like structure was measured. Therefore, the biomechanical parameters results of MSCs group of this study, although seems not to be beneficial (with the exemption of cartilage thickness), definitely is superior to subchondral bone. Proinflammatory cytokines like TNF-a and IL-1b have prominent role in blood-induced joint damage [54]. TNF-a showed a reasonable decrease in the MSCs in comparison to the other groups and IL-1b was higher than Con, but slightly lower than Con© 2015 John Wiley & Sons Ltd
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ConHem 10
ConHem 38
MSCs
Control
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Fig. 5. Electromicrograph of the hyaline cartilage of different groups. Bars: 100 lm.
Fig. 6. Hole in the medial plateau of tibia bone of ConHem38 group (left), Repaired hole in mesenchymal stem cells group (right).
Hem38. The role of anti-inflammatory cytokines as specific therapies has gained attention in preventing BIJD in parallel with clotting factor replacement [27,55,56]. Il-4 slightly increased in MSCs in comparison to its control group and could reveal that MSCs are capable of gently touching the anti-inflammatory condition of the EH damaged joint. Greish et al., found that TNF-a and Il-1b decreased significantly in © 2015 John Wiley & Sons Ltd
MSCs group in comparison to control in complete Freunds adjuvant induced arthritis of rats hind paw and IL-10 increased significantly. However, the cytokine assays performed in the rat serum and the arthritis model was not confined to a joint space like the present study [57]. The SEM results showed MSCs could regenerate chondrocytes despite the fact that the force and stiffness of the newly formed cartilage were low. One explanation is that the chondrocytes did not have plenty of time to produce more cartilage matrix to be able to withstand loading. There might have been a possibility for further improvement of the mechanical properties of the cartilage with a longer follow up. The scanning electron micrograph of ConHem38 depicts the breakdown of cartilage structure and confirms the long-term potential of blood in cartilage damage [29,30]. This amount of arthropathy in an animal model with normal coagulation conveys the Haemophilia (2015), 21, 693--701
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importance of prophylaxis and the need of new kinds of treatment like MSCs therapy in PWH. Safety is an important prerequisite for MSCs therapy, particularly in orthopaedic field which is not life threatening. The BM-derived MSCs were checked for tumour development and infections in 41 patients received 45 transplantation and neither tumour nor infections were observed for a mean 75 (5–137) months of following up [58]. Based on Peeters et al. review on the safety of IA cell-therapy with cultureexpanded stem cells, this application reported to be safe [59]. Moreover, age-related biological characterization of MSCs in a human articular cartilage study showed spontaneous osteogenic differentiation besides lower chondrogenic differentiation only in elderly patients. As HA predominantly happens in younger ages, It would be expected to find more reasonable results through MSCs IA injection [60]. In conclusion, this study shows that haemarthrosis if left untreated could lead to severe cartilage damage. MSCs IA injection after EH could reduce
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friction, slightly decreasing the inflammatory and increasing the anti-inflammatory conditions. Although chondrocytes did not still have proper mechanical strength, they regenerated remarkably. Therefore, MSCs IA injection is promising for chondrocyte loss in experimental HA. Further studies are definitely required.
Acknowledgements The authors would like to thank Prof Abron for his support in MSCs culture. This study was supported by a grant from presidency of the Islamic Republic of Iran, vice-presidency for science and technology. The study sponsor had no involvement in the research procedure or writing the manuscript or in the decision to submit the manuscript for publication.
Disclosures The authors stated that they had no interests which might be perceived as posing a conflict or bias.
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