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Volume 5 • Issue 11 • 1000314J Stem Cell Res TherISSN: 2157-7633 JSCRT, an open access journal

Open AccessResearch Article

Miki et al., J Stem Cell Res Ther 2015, 5:11 DOI: 10.4172/2157-7633.1000314

AbstractObjective: The purpose of this study was to evaluate effectiveness of intra-articular injection of synovium-derived

mesenchymal stem cells (SMSCs) and hyaluronic acid (HA) for the treatment of articular cartilage defects in a canine model.

Methods: Forty-eight knees of 24 beagle dogs were randomly assigned to 16 groups (n=3) according to both the number of injected SMSCs (0.5×105 cells, 5×106 cells, 5×107 cells) and the concentration of HA (0%, 0.01%, 0.1%, 0.5%). A partial-thickness cartilage defect was created in the medial femoral condyle under arthroscopy. After seven weeks, autologous SMSCs with or without 1 ml HA were percutaneously injected into the injured knee. In the control group, 1 ml saline was injected. Twelve weeks after the injection, evaluation was performed using the International Cartilage Repair Society (ICRS) visual assessment scale and the modified O’Driscoll histological score.

Results: The mean ICRS visual assessment scale and the mean modified O’Driscoll histological score were 1.0 ± 0.00 and 11.0 ± 0.00 in the control group. On the other hand, the mean ICRS visual assessment scale and the mean modified O’Driscoll histological score were 7.3 ± 3.21 and 30.7 ± 7.23 in the group injected with 5×106 cells and 0.01% HA.

Conclusion: Intra-articular injection of SMSCs with HA may be effective for stimulating articular cartilage repair. Our results suggest that there is an ideal combination of the number of SMSCs and the concentration of HA for promoting articular cartilage repair.

Intra-Articular Injection of Synovium-Derived Mesenchymal Stem Cells with Hyaluronic Acid Can Repair Articular Cartilage Defects in a Canine ModelShinya Miki*, Masato Takao, Wataru Miyamoto, Takashi Matsushita and Hirotaka KawanoDepartment of Orthopaedic Surgery, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi, Tokyo, 173-8605, Japan

*Corresponding author: Shinya Miki, M.D., Department of Orthopaedic Surgery, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi, Tokyo, 173-8605, Japan, Tel: 81-3-3964-4097; Fax: 81-3-3964-1211; E-mail: [email protected]

Received October 14, 2015; Accepted November 03, 2015; Published November 05, 2015

Citation: Miki S, Takao M, Miyamoto W, Matsushita T, Kawano H (2015) Intra-Articular Injection of Synovium-Derived Mesenchymal Stem Cells with Hyaluronic Acid Can Repair Articular Cartilage Defects in a Canine Model. J Stem Cell Res Ther 5: 314. doi:10.4172/2157-7633.1000314

Copyright: © 2015 Miki S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Keywords: Cartilage defect; Mesenchymal stem cells; Synovium;Hyaluronic acid; Intra-articular injection

IntroductionArticular cartilage has only limited intrinsic ability to heal itself.

Under physiological circumstances, articular cartilage injuries rarely heal spontaneously and usually progress to osteoarthritis [1]. Various treatment methods, including bone marrow stimulation, osteochondral transplantation, autologous chondrocyte implantation, and tissue engineering, have been developed to repair articular cartilage defects [2-4]. However, the results are still controversial and the gold standard remains to be established.

Mesenchymal stem cells (MSCs) transplantation is attracting attention because MSCs are able to migrate to injured tissue in response to signaling pathways [5-7] and have the potential for multipotent differentiation [8,9]. Of MSCs derived from various sites, synovium-derived MSCs (SMSCs) are regarded as a promising therapeutic cell type for cartilage repair because they have higher capacity for chondrogenic differentiation than MSCs derived from other locations [10-12]. Successful results of the in vivo treatment for articular cartilage defects using SMSCs have been reported [13-15], but some problems still remain. The first is that an open arthrotomy is required to create cartilage defects. This incision required for this procedure causes intra-articular bleeding, which may affect the results of the studies [16]. The second problem is that invasive surgery, such as open arthrotomy or arthroscopy, is required for cell transplantation. To resolve these problems, we created cartilage defects under arthroscopy to minimize bleeding from the incision [17]. In addition, we transplanted SMSCs via intra-articular injection as a less invasive method. Recently, several studies have shown that intra-articularly injected MSCs can migrate to cartilage defects in response to signaling pathways and repair articular cartilage defects [5,6].

Intra-articular injection of hyaluronic acid (HA) has been widely used in the treatment of osteoarthritis [18] because it has chondroprotective and cytoprotective effects [19,20]. In addition, several animal studies have suggested that intra-articular injection of MSCs with HA is a feasible and promising minimally invasive method for cartilage repair [21,22]. Based on these studies, we hypothesized that intra-articular injection of SMSCs and HA could repair articular cartilage defects. Accordingly, the purpose of the present study was to evaluate the effectiveness of intra-articular injection of autologous SMSCs and HA for the treatment of articular cartilage defects in a canine model.

Materials and MethodsAnimal model

Forty-eight knees of 24 adult male beagle dogs were used in this study. All dogs were aged between 11 and 13 months and weighed between 9.0 and 11.6 kg at the time of surgery. The study was approved by the Institutional Animal Care and Use Committee and conducted in compliance with the Teikyo University Committee for Laboratory

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Animal Research Guidelines. The dogs were randomly assigned to 16 groups (n=3) according to both the number of injected SMSCs (0.5×105 cells, 5×106 cells, 5×107 cells) and the concentration of HA (0%, 0.01%, 0.1%, 0.5%). Neither SMSCs nor HA were injected in the control group.

Surgical procedure

Surgery was aseptically performed with animals under general anesthesia. Anesthesia was induced via intramuscular ketamine at 15 mg/kg and maintained with inhalational halothane fewer than 100% oxygen. Knee arthroscopy was performed with the use of two portals. A partial-thickness cartilage defect (4 mm in diameter) was created in the medial femoral condyle by use of a punch, and the synovium was harvested simultaneously. Care was taken not to breach the subchondral bone. The wound was sutured with No. 3-0 Vicryl (Ethicon, Somerville, NJ, USA). The dogs were allowed to move freely within their cages after surgery.

Isolation and expansion of SMSCs

The harvested synovium was minced, washed in phosphate-buffered saline (PBS) solution, and then digested with 0.02% collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 2 hr at 37°C. After being filtered through a 70 µm nylon filter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), the digested cells were washed and resuspended in culture medium (STK1 and STK2; DS Pharma Biomedical Co., Osaka, Japan) supplemented with 1% antibiotic/antimycotic solution (Gibco, NY, USA). Then, 5×104 cells were plated in 150 mm diameter culture dishes (Nunc, Roskilde, Denmark) at 37°C in a humidified 5% carbon dioxide atmosphere. The medium was changed after 3 days to allow cell adhesion and to remove nonadherent cells. SMSCs in passage 3 were used for the experiments in this study.

Multipotent differentiation of SMSCs

Adipogenic differentiation was induced by culturing SMSCs for 21 days in adipogenic medium (Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 1 µM dexamethasone (Sigma), 0.5 mM isobutylmethylxanthine (Sigma), 10 µg/ml insulin, and 100 µM indomethacin (Wako)) and assessed using oil red O stain. Osteogenic differentiation was induced by culturing SMSCs for 21 days in osteogenic medium (DMEM containing 10% FBS, 1 nM dexamethasone, 10 mM β-glycerol phosphate (Wako), and 50 µM ascorbic acid) and assessed using alizarin red stain. Chondrogenic differentiation was induced using the micromass culture technique: 2.5×105 cells were pelleted into a 15 ml polypropylene tube (Becton-Dickinson) and cultured in chondrogenic medium (DMEM containing 1% FBS, 50 µg/ml ascorbic acid, 1000 ng/ml transforming growth factor-β3 (R&D Systems, Inc., Minneapolis, MN, USA), and 6.25 µg/ml insulin) for 21 days. The pellets were gently embedded in paraffin and cut into 5 µm sections. Chondrogenesis was confirmed by toluidine blue stain [23].

Intra-articular injection of SMSCs

Seven weeks after the creation of the cartilage defects, autologous SMSCs with or without 1 ml HA were percutaneously injected into the injured knee joint under ultrasound control in each group. In the control group, 1 ml saline was injected instead of HA. The dogs were allowed to move freely within their cages after the injection.

Macroscopic analysis

Twelve weeks after the injection, all dogs were sacrificed by means of sodium pentobarbital (Kyoritu Pharma Co., Tokyo, Japan) overdose.

The knee joints were opened and the macroscopic appearance of the defects was assessed by two independent researchers in a blinded manner using the International Cartilage Repair Society (ICRS) visual assessment scale [24,25]. The details of the scoring system are listed in Table 1.

Histological analysis

After macroscopic examination, the femurs were fixed in 10% formalin for 5 days, decalcified with ethylene diamine tetra-acetate (EDTA) for 7 days, dehydrated via a graded series of ethanol solutions, and then embedded in paraffin. Sagittal sections (4 µm thick) were stained with hematoxylin and eosin and safranin O. Each slice was assessed by two independent researchers in a blinded manner using the modified O’Driscoll histological score [26]. This scale yields a score from 11 to 45, with high scores indicating good repair tissue that approximates normal hyaline cartilage. The details of the scoring system are listed in Table 2. For immunohistochemical analysis, sections were immunostained with monoclonal antibodies against type II collagen (Collagen Staining Kit 2.0; Chondrex, Redmond, WA, USA). Immunohistochemical sections were pretreated with 1% hydrogen peroxide and incubated for 30 minutes with 2% bovine testicular hyaluronidase dissolved in PBS at 25°C, followed by 1 hr incubation with monoclonal antibody solution at room temperature. After washing with PBS, the sections were incubated with diluted streptavidin peroxidase solution for 1 h at room temperature.

ResultsMultipotent differentiation of SMSCs

Synovium-derived colony-forming cells differentiated into adipocytes and chondrocytes and were calcified when cultured in their respective differentiation media (Figures 1A-1C).

Categories Score

Degree of defect repair

In level with surrounding cartilage 475% repair of defect depth 350% repair of defect depth 225% repair of defect depth 10% repair of defect depth 0

Integration to border zone

Complete integration with surrounding cartilage 4Demarcating border <1 mm 33/4 of graft integrated, 1/4 with a notable border >1 mm width 21/2 of graft integrated with surrounding cartilage, 1/2 with a notable border >1 mm 1

From no contact to 1/4 of graft integrated with surrounding cartilage 0

Macroscopic appearance

Intact smooth surface 4Fibrillated surface 3Small, scattered fissures or cracs 2Several, small or few but large fissures 1Total degeneration of grafted area 0

Overall repair assessment

Grade I: normal 12Grade II: nearly normal 11-8Grade III: abnormal 7-4Grade IV: severly abnormal 3-1

Table 1: ICRS visual assessment scale.


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Variable Comment Score

Tissue Morphology (Ti) Mostly hyaline cartilage 4

Mostly fibrocartilage 3

Mostly non-cartilage 2

Exclusively non-cartilage 1

Matrix staining (Matx) None 1

Slight 2

Moderate 3

Strong 4

Structural integrity (Stru) Severe disintegration 1

Cysts or disruption 2

Beginning of columnar organization of chrondocytes 4

Normal, similar to healthy mature cartilage 5

Chondrocyte clustering in implant (Clus) 25-100% of the cells clustered 1

<25% of the cells clustered 2

No clusters 3

Intactness of the calcified layer, formation of tidemark (Tide) <25% of the calcified layer intact 1

25-49% of the calcified layer intact 2



Complete intactness of the calcified cartilage layer 5

Subchondral bone formation (Bform) No formation 1

Slight 2

Strong 3

Histological appraisal of surface architecture (SurfH) Severe fibrillation of disruption 1

Moderate fibrillation or irregularity 2

Slight fibrillation or irregularity 3

Normal 4

Histological appraisal defect filling (FilH) <25% 1

26-50% 2

51-75% 3

76-90% 4

91-110% 5

Lateral integration of implanted material (Latl) Not bonded 1

Bonded at one hand/partially both ends 2

Bonded at both sides 3

Basal integration of implanted material (Basl) <50% 1

50-70% 2

70-90% 3

91-100% 4

Inflammation (InfH) No inflammation 1

Slight inflammation 3

Strong inflammation 5

Maximum total score 45

Table 2: Modified O’Driscoll histological score.


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Macroscopic analysis

The results of ICRS visual assessment scale were illustrated in Table 3. Macroscopic evaluation showed that no healing occurred in the control group treated with lactated Ringer’s solution. The defect margins were clearly distinguishable, and there was no reparative tissue filling (Figure 2A). The mean ICRS visual assessment scale was 1.0 ± 0.00. On the other hand, the typical macroscopic appearance of the group injected with 5×106 cells and 0.01% HA indicated marked improvement in the defect filling compared with the control group. The reparative tissue showed good integration at the margins with a smooth surface (Figure 2B). The mean ICRS visual assessment scale was 7.3 ± 3.21.

Histological analysis

The results of modified O’Driscoll histological score were illustrated in Table 4. Histological evaluation showed that no healing occurred in the control group treated with lactated Ringer’s solution. Defects were not filled with any reparative tissue (Figures 3A-3F). The mean modified O’Driscoll histological score was 11.0 ± 0.00. On the other hand, the typical histological appearance of the group injected with 5×106 cells and 0.01% HA indicated marked improvement in the defect filling compared with the control group, as seen in the macroscopic evaluation. Defects were filled with hyaline-like cartilage with good

Figure 1: Multipotentiality of synovium-derived mesenchymal stem cells (SMSCs). A: Cartilage pellets were sectioned and stained with toluidine blue. B: Adipocytes were stained with oil red O. C: Calcifications were stained with alizarin red. (Scale bars=100 µm).

Figure 3: Histological observation of the defects in the two groups at 12 weeks after the injection. The regions in the small boxes are magnified in the figures below. A–F: control group; G–L: the group injected with 5 × 106 cells and 0.01% HA; A, B, G, H: hematoxylin and eosin staining, C, D, I, J: safranin O staining, E, F, K, L: immunochemical staining for type II collagen. (Scale bars=2 mm in the upper row/200 µm in the lower row).

Number of SMSCsConcentration of HA 0 5x105 5x106 5x107

0% 1.0±0.00 1.7±0.58 2.0±1.00 2.7±0.580.01% 3.3±2.08 3.0±0.00 7.3±3.21 5.3±4.930.1% 4.0±1.00 2.3±0.58 3.7±0.58 2.3±0.580.5% 3.3±2.08 2.0±0.00 3.0±1.00 2.7±0.58

Table 3: Results of macroscopic observation.

Number of SMSCsConcentration of HA 0 5x105 5x106 5x107

0% 11.0±0.00 12.3±1.15 15.7±3.79 15.0±2.000.01% 18.7±0.58 18.3±3.21 30.7±7.23 22.3±12.740.1% 17.0±2.65 17.0±2.00 18.7±2.52 14.0±3.460.5% 21.7±9.45 18.0±1.73 15.3±0.58 16.3±0.58

Table 4: Results of histological observation.

Figure 2: Macroscopic observation of the defects at 12 weeks after the injection. The black arrows show the defects or repaired tissues. A: control group; B: the group injected with 5 × 106 cells and 0.01% HA. (Scale bars=4 mm).


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integration, thickness, and a smooth surface. With safranin O staining, there was marked proteoglycan accumulation in the deep layers, and there was a considerable amount of chondrocyte-like cells that were well-organized in columns. Immunochemical staining for type II collagen showed that it was distributed throughout the entire reparative tissue (Figures 3G-3L). The mean modified O’Driscoll histological score was 30.7 ± 7.23.

Complications

There was no evidence of complications in any case.

DiscussionIn the present study, we showed that intra-articular injection of

SMSCs with HA could help to repair articular cartilage defects. In addition, the results of the control group suggest that a partial-thickness cartilage defect does not have the potential to spontaneously heal itself.

In most previous studies using MSCs, open arthrotomy was performed to create cartilage defects and permit cell transplantation. However, the incision causes intra-articular bleeding. Roosendaal et al. [16] reported that intra-articular blood had a direct irreversible harmful effect on cartilage, via mononuclear cells and erythrocytes. Therefore, in this study, we tried to reduce the harmful effect of intra-articular bleeding by creating cartilage defects under arthroscopy and by transplanting the SMSCs by intra-articular injection.

Recently, several studies have suggested that intra-articular injection of bone marrow-derived MSCs (BMSCs) with HA is a feasible and promising minimally invasive method for cartilage repair. Murphy et al. [22] reported reduced degeneration of the articular cartilage in a caprine model of osteoarthritis using injection of BMSCs and HA. Lee et al. [21] reported successful repair results in their study of a partial-thickness cartilage defect in a porcine model using injection of BMSCs and HA. In contrast to these studies, we used SMSCs instead of BMSCs. When the chondrogenic potential of MSCs derived from bone marrow, adipose tissue, synovium, and skeletal muscle were compared in vitro and in vivo, the superior chondrogenesis of SMSCs was shown [10-12].

The present study found that the group injected with 5×106 cells and 0.01% HA showed the best results, suggesting that the relationship between the number of injected cells and the concentration of HA may be important for articular cartilage repair. There is a noticeable lack of information on the effect of the specific combination of the number of MSCs and the concentration of HA on articular cartilage repair. Suzuki et al. [15] reported that transplantation of aggregates of SMSCs at high density failed to repair cartilage due to cell death and nutrient deprivation of SMSC aggregates, and that transplantation of aggregates of SMSCs at relatively low density achieved successful cartilage repair. They concluded that 2.5×106 SMSCs per 25 mm2 defect (1.0×105 SMSCs per 1 mm2 defect) were needed for better cartilage repair. In our study, 5.0×106 SMSCs per 16 mm2 (3.1×105 SMSCs per 1 mm2 defect) achieved the best results. The minor difference in the results may be due to the transplantation method. Suzuki et al. [15] performed open arthrotomy and transplanted SMSCs directly into the defect site. On the other hand, we transplanted SMSCs by intra-articular injection.

The chondroprotective and cytoprotective effects of HA have been shown in many studies. In osteoarthritis models, cartilage degeneration was reduced by HA [27,28]. Liu et al. [19] reported that HA protected chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Grishko et al. [29] reported that pretreatment of chondrocytes with HA can decrease mitochondrial DNA damage,

preserve ATP levels, and increase cell viability. Moreover, Yoshioka et al. [30] reported that synovial cell and chondrocyte migration were increased with HA. However, high-concentration HA may not be effective for articular cartilage repair. Erickson et al. [31] reported that MSCs seeded in low macromer concentration (1%) HA gels achieved better cartilage matrix distribution than those seeded in high macromer concentration (3% and 5%) HA gels. In our study, the low HA concentration (0.01%) groups tended to achieve better results than the other HA concentration (0.1% and 0.5%) groups. Therefore, we postulate that the high viscosity of high-concentration HA might prevent cell migration. However, the relation between the concentration of HA and the migration ability of cells is not fully understood. Our results suggest that, for promoting articular cartilage repair, there is an ideal relationship between the number of MSCs and the concentration of HA. However, further studies are needed to determine the ideal conditions.

There are some limitations to our study. The first limitation is the small sample size, which meant that statistical analysis could not be performed. Thus, although the group injected with 5×106 cells and 0.01% HA showed the best results, the findings may not be meaningful. The second limitation is that the cellular origin of the repaired cartilage after cell transplantation was not well defined. In future studies, cell labeling should be performed to confirm that the injected SMSCs were found in the reparative tissue. The third limitation is that there were large inter-individual differences in the visual assessment and histological scores. Although the group injected with 5×106 cells and 0.01% HA showed the best results, the individual ICRS visual assessment scale scores were 5, 6, and 11 and the modified O’Driscoll histological scores were 26, 27, and 39. We speculate that there may be other factors, such as aberrant cytokines, that affect the cartilage healing process. Further studies are needed to determine the cause of such inter-individual differences. In spite of these limitations, the present study demonstrated a novel treatment for articular cartilage defects. This method has a possibility to repair articular cartilage defects and to restore its functions with minimal invasion.

ConclusionsIntra-articular injection of SMSCs with HA may be an effective

method for stimulating articular cartilage defect repair. Our results suggest that there is an ideal combination of the number of SMSCs and the concentration of HA for promoting articular cartilage repair.

Acknowledgements

We would like to thank Yuko Hotta for providing technical assistance with histochemical staining, and Azusa Seki for providing technical assistance with animal anesthesia and postoperative care.

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