Transplantation of Autologous Synovial Mesenchymal Stem Cells Promotes Meniscus Regeneration in Aged Primates

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1 Transplantation of Autologous Synovial Mesenchymal Stem Cells Promotes Meniscus Regeneration in Aged Primates Shimpei Kondo, 1 Takeshi Muneta, 1 Yusuke Nakagawa, 1,2 Hideyuki Koga, 1 Toshifumi Watanabe, 3 Kunikazu Tsuji, 3 Shinichi Sotome, 4 Atsushi Okawa, 5 Shinji Kiuchi, 6 Hideo Ono, 6 Mitsuru Mizuno, 2 Ichiro Sekiya 2 1 Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, 2 Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan, 3 Department of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo , Japan, 4 Department of Orthopaedic Research and Development, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, 5 Department of Orthopaedic and Spinal Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, 6 Yaesu Clinic, Tokyo, Japan Received 29 October 2015; accepted 17 February 2016 Published online 24 April 2017 in Wiley Online Library (wileyonlinelibrary.com). DOI /jor ABSTRACT: Transplantation of aggregates of synovial mesenchymal stem cells (MSCs) enhanced meniscus regeneration in rats. Anatomy and biological properties of the meniscus depend on animal species. To apply this technique clinically, it is valuable to investigate the use of animals genetically close to humans. We investigated whether transplantation of aggregates of autologous synovial MSCs promoted meniscal regeneration in aged primates. Chynomolgus primates between 12 and 13 years old were used. After the anterior halves of the medial menisci in both knees were removed, an average of 14 aggregates consisting of 250,000 synovial MSCs were transplanted onto the meniscus defect. No aggregates were transplanted to the opposite knee for the control. Meniscus and articular cartilage were analyzed macroscopically, histologically, and by MRI T1rho mapping at 8 (n ¼ 3) and 16 weeks (n ¼ 4). The medial meniscus was larger and the modified Pauli s histological score for the regenerated meniscus was better in the MSC group than in the control group in each primate at 8 and 16 weeks. Mankin s score for the medial femoral condyle cartilage was better in the MSC group than in the control group in all primates at 16 weeks. T1rho value for both the regenerated meniscus and adjacent articular cartilage in the MSC group was closer to the normal meniscus than in the control group in all primates at 16 weeks. Transplantation of aggregates of autologous synovial MSCs promoted meniscus regeneration and delayed progression of degeneration of articular cartilage in aged primates. This is the first report dealing with meniscus regeneration in primates. ß 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35: , Keywords: mesenchymal stem cells; synovium; meniscus; regeneration; primate The meniscus functions as a cushion to minimize the stress on articular cartilage in the knee. For meniscus injury, the meniscus should be preserved as much as possible in order to delay progression of degeneration of articular cartilage. 1,2 After meniscectomy or meniscal degeneration, transplantations of the meniscal graft or artificial meniscus have been attempted 3,4 ; however, the outcome of the transplantations is still controversial. 5,6 A new treatment to regenerate the meniscus is needed. The synovium plays pivotal roles during the natural course of meniscal healing and contains mesenchymal stem cells (MSCs) with high chondrogenic potential. 7,8 We previously reported that intra-articular injection of synovial MSCs promoted meniscus regeneration after meniscectomy in rats and rabbits. 9,10 Transplantation of aggregates of synovial MSCs also enhanced meniscus regeneration in rats. 11 Furthermore, placing synovial MSC suspension on the meniscus lesion for 10 min promoted meniscus regeneration after meniscectomy and healing after meniscus repair in pigs. 12,13 Anatomy and biological properties of the meniscus depend on animal species In order to apply the Conflicts of interest: None. Grant sponsor: The Japan Agency for Medical Research and Development (AMED); Grant sponsor: The Japan Society for the Promotion of Science; Grant number: Correspondence to: Ichiro Sekiya (T: þ ; F: þ ; sekiya.arm@tmd.ac.jp) # 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. findings obtained by basic research to clinical situations, it is valuable to investigate the use of animals genetically close to humans. In this study, we investigated whether transplantation of synovial MSCs promoted meniscal regeneration in aged primates. This is the first report dealing with meniscus repair/regeneration in primates. MATERIALS AND METHODS Animals All experiments were conducted in accordance with the institutional guidelines for the care and use of experimental animals at Tokyo Medical and Dental University. We used seven skeletally mature cynomolgus macaques from 12 to 13 years of age, weighing kg, 8.3 kg on average. All primates had free access during the study period to food and water in a post-operative care cage. Cell Isolation and Culture Under general anesthesia by intramuscular injection of 2.5 mg/kg ketamine and 0.25 mg/kg medetomidine, a 3 cm straight incision was made on the anterior side of the both knees, each knee was approached through an anteromedial parapatellar arthrotomy. Approximately 300 mg of synovial tissue was harvested from the infrapatellar fat pad of both knees, including the control side, of all seven primates. Synovial tissue with the underlying connective tissue was digested in 3 mg/ml collagenase (Sigma Aldrich Japan, Tokyo, Japan) in a-minimal essential medium (a-mem; Invitrogen, Carlsbad, CA) at 37 C for 3 h, and filtered through a 70-mm nylon filter (BD Biosciences, Franklin Lakes, NJ). The nucleated cells were plated in 150-cm 2 culture dishes (Nunc, Rochester, NY) at cells/dish in 1274

2 MENISCUS REGENERATION BY SYNOVIAL MSCs IN PRIMATES 1275 complete medium (a-mem supplemented with 10% fetal bovine serum [FBS], 100 U/L penicillin, 100 mg/ml streptomycin, and 250 ng/ml amphotericin B [all from Invitrogen]) in 150-cm 2 culture dishes (Nunc), and then cultured for 14 days at 37 C, 5% CO 2 with saturated humidity. The adherent cells were harvested with 0.25% trypsin-edta (Invitrogen) as passage 0. The cells were plated in 150-cm 2 culture dishes at cells/dish, and cultured for 11 days as passage 1. Passage 1 cells were used for further studies. Colony-Forming Unit Assay We plated 500 cells were plated in 60-cm 2 dishes, cultured them in complete medium for 14 days, and stained them Figure 1. Characteristics of primate synovial MSCs. (A) Cell morphology, colony formation, and multipotentiality. (B) Total cell number obtained per primate at passages 0 and 1. The proliferation of synovial MSCs derived from seven primates was evaluated.

3 1276 KONDO ET AL. with 0.5% crystal violet in methanol for 5 min in order to observe cell colonies. In Vitro Differentiation Assay For chondrogenesis, synovial MSCs were collected at the bottom of a 15-ml polypropylene tube (BD Biosciences) by centrifugation at 450g for 10 min. The pellets were cultured for 21 days in chondrogenesis medium consisting of high-glucose Dulbecco s modified Eagle s medium (Invitrogen) supplemented with 1 mg/ml bone morphogenetic protein (BMP)-7 (provided from Stryker Biotech, Hopkinton, MA), 10 ng/ml transforming growth factor (TGF)-b3 (R&D Systems, Minneapolis, MN), 100 nm dexamethasone (Sigma Aldrich, St Louis, MO), 50 mg/ml ascorbate-2-phosphate, 40 mg/ml proline, 100 mg/ml pyruvate, and 1:100 diluted ITS þ Premix (6.25 mg/ml insulin, 6.25 mg/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid; BD Biosciences). For adipogenesis, synovial MSCs were cultured for 21 days in adipogenic medium consisting of complete medium supplemented with 100 nm dexamethasone, 0.5 mm isobutylmethylxanthine (Sigma Aldrich) and 50 mm indomethacin (Wako, Tokyo, Japan). The adipogenic cultures were fixed in 4% paraformaldehyde, and then stained with fresh oil red-o solution. For calcification, synovial MSCs were cultured for 21 days in calcification medium consisting of complete medium supplemented with 1 nm dexamethasone, 20 mm b-glycerol phosphate (Wako), and 50 mg/ml ascorbate-2-phosphate (Sigma Aldrich). The cells were fixed in 4% paraformaldehyde, and then stained with 0.5% alizarin red solution. Multilineage differentiation was evaluated in synovial MSCs derived from only one primate. Aggregates of Synovial MSCs We suspended 250,000 synovial MSCs in 35 ml complete culture medium, and plated them on an inverted culture dish lid. Then, the lid was inverted and placed on a culture dish. The cells were cultured at 37 C with 5% humidified CO 2 for 3 days in hanging drops. Meniscectomy A straight incision was made on the anterior side of the bilateral knees, each knee was approached through a medial parapatellar arthrotomy, and the anterior insertional ligament of the medial meniscus was transected. Then, the Figure 2. Transplantation of aggregates of primate synovial MSCs. (A) Experimental set up. (B) Drops hanging on the cover of dish. An aggregate consisting of 25,000 MSCs is indicated with an arrow. (C) Meniscectomy and transplantation of aggregates of MSCs. (i) The medial meniscus was dislocated anteriorly. (ii) The anterior half of the meniscus was removed. (iii) The aggregates (indicated with white arrows) were transplanted. MFC, medial femoral condyle; MM, medial meniscus.

4 MENISCUS REGENERATION BY SYNOVIAL MSCs IN PRIMATES 1277 anterior half of the medial meniscus was dislocated and resected at the level of the medial collateral ligament. Transplantation of Aggregates Four weeks after synovium was harvested, the aggregates of autologous synovial MSCs were placed in the meniscus defects and were kept in place by gravity and surface tension. The number of aggregates was 7 20, 14 on average, dependent on yields of synovial MSCs at passage 1. No aggregates were transplanted in the opposite knee. After waking up from anesthesia, the primates were allowed to walk freely without fixation in each group. The knee joints were evaluated at 8 weeks (n ¼ 3) and 16 weeks (n ¼ 4). To remove individual variability, both sides of knees in the same primate were compared. Macroscopic Observation The meniscus was carefully removed and macroscopic pictures were taken using an Olympus MVX 10 (Olympus, Tokyo, Japan). Quantification for the size of the regenerated meniscus was performed using the software ImageJ (National Institutes of Health, Bethesda, MD). Histological Analysis Specimens of medial menisci and medial femoral condyles were fixed separately in 4% paraformaldehyde for 7 days, Figure 3. Macroscopic analysis of the regenerated meniscus. (A) Macroscopic observation of the medial menisci. Regenerated area is surrounded with a yellow dotted line. To remove individual variability, the regenerated menisci in the MSC and control groups of the same primate are shown in the same row. (B) Quantification of area for medial menisci.

5 1278 KONDO ET AL. Figure 4. Histological analysis of the regenerated meniscus. (A) Radial sections for the intact and regenerated menisci stained with safranin-o. To remove individual variability, the regenerated menisci in the MSC and control groups of the same primate are shown in the same row. (B) Quantification of histology evaluated by a modified Pauli s scoring system (Supplementary Table S1). 12,17 and then dehydrated with a gradient ethanol series. In order to section finely, the specimens were decalcified with 20% EDTA (ph 7.4) at room temperature for 21 days. The specimens were embedded in paraffin and sectioned into slices 5-mm thick, radially for the meniscus and sagittally for the condyles. The sections were stained with safranin-o/fast green. To quantify the regeneration of the meniscus, a modified Pauli s scoring system was used (Supplementary

6 MENISCUS REGENERATION BY SYNOVIAL MSCs IN PRIMATES 1279 Table S1). 12,17 To quantify the extent of articular cartilage degeneration, Mankin s scoring system was used, on a scale of 0 14 points (Supplementary Table S2). 18 Magnetic Resonance Imaging Evaluation MRI analysis was performed with 3.0 T imager (Achieva; Philips Medical Systems, Andover, MA) and a coil for the wrist (Phillips Medical System). The morphology of the meniscus and other knee structures were assessed using a fat-suppressed three-dimensional multi-echo gradient recalled echo sequence (3D mgre). A sagittal 3D T1rho imaging was also taken. For setting the regions of interest (ROIs) of the medial meniscus, segmentation of the anterior part of the medial meniscus was performed referring to 3D mgre images. For setting the ROIs of the medial femoral condyle, segmentation of the medial femoral condyle between the anterior horn of the meniscus and its posterior horn, which were contacting regions of femoral and tibial cartilage while standing, was performed referring to 3D mgre images. Quantification of MRI analysis was performed for the regenerated meniscus and the cartilage of the medial femoral condyle using the sagittal T1 rho mapping. The T1 rho values were calculated with Ziostation2 software (Ziosoft, Tokyo, Japan). 19 RESULTS Properties of Primate Synovial MSCs We observed colony formation 14 days after 500 synovial nucleated cells were cultured in a 60-cm 2 dish (Fig. 1A). The colony-forming cells differentiated into adipocytes positively stained with oil red-o and mineralized tissue positively stained with alizarin red. The pellet of the colony-forming cells also differentiated into cartilage positively stained with toluidine blue. The yields of synovial MSCs per primate were 1.0 million in six primates among seven at passage 0, and 25.5 million at maximum, 2.0 million at minimum, and 7.8 million on average at passage 1 (Fig. 1B). The fold increase between passages 0 and 1 was (mean standard deviation). Histologically, the regenerated meniscus was slightly stained with safranin-o in the MSC group, while hardly stained in the control group at 8 weeks (Fig. 4A). The regenerated meniscus was positively stained with safranin-o in the MSC group, while still hardly stained in the control group at 16 weeks. The modified Pauli s histological score was better in the MSC group than in the control group in all three primates at 8 weeks and in all four primates at 16 weeks (Fig. 4B). T1rho mapping image for the anterior medial meniscus in the MSC group appeared to be closer to that in the intact meniscus than that in the control group (Fig. 5A). T1rho value was shorter in the MSC group than in the control group in all four primates (Fig. 5B). Prevention of Cartilage Degeneration by Aggregates of Synovial MSCs Histologically, articular cartilage was degenerated in both groups in all four primates at 16 weeks (Fig. 6A). Mankin s score for histology was better in the MSC group than in the control group in all four primates at 16 weeks (Fig. 6B). T1rho value for the medial femoral cartilage was shorter in the MSC group than in the control group in all four primates (Fig. 6C and D). DISCUSSION In this study, we demonstrated that colony-forming cells derived from synovium of cynomolgus macaques possessed multipotentiality, a character of MSCs. Transplantation of synovial MSCs promoted meniscus Appearance of Aggregates of Synovial MSCs Primate synovial MSCs were aggregated using the hanging drop culture (Fig. 2A). The aggregate, consisting of 250,000 MSCs, became 1 mm in diameter 3 days after hanging drop culture (Fig. 2B). The aggregate was not easily broken by manipulation. Meniscus Regeneration by Aggregates of Synovial MSCs After meniscectomy, 7 20 aggregates were placed on the sites of meniscal defects where they were able to remain through gravity and surface tension (Fig. 2C). Macroscopically, the anterior half of the medial meniscus, where the native meniscus was previously removed, appeared to regenerate both in the MSC and control groups. The medial meniscus was larger in the MSC group than in the control group in all three primates at 8 weeks (Fig. 3A and B). The medial meniscus was still larger in the MSC group than in the control group in all four primates at 16 weeks. Figure 5. MRI analysis of the regenerated meniscus. (A) Representative sagittal MRI of gradient recall echo (GRE) and T1rho mapping at the center of the regenerated meniscus. (B) Evaluation of T1rho values for the regenerated meniscus.

7 1280 KONDO ET AL. Figure 6. Histological and MRI analyses of cartilage at the medial femoral condyle. (A) Radial sections stained with safranin-o. (B) Quantification of histology evaluated by Mankin s scoring system. 18 (C) Representative sagittal MRI of GRE and T1rho mapping at the center of the articular cartilage of the medial femoral condyle. (D) Evaluation of T1rho value for the medial femoral condyle. regeneration and delayed progression of degeneration of articular cartilage in primates. This is the first report demonstrating the usefulness of synovial MSCs for meniscus regeneration in primates. We previously reported that transplantation of synovial MSCs promoted meniscus regeneration after the anterior half of the medial menisci was resected in rats, rabbits, and minipigs The duration to obtain

8 MENISCUS REGENERATION BY SYNOVIAL MSCs IN PRIMATES 1281 meniscus regeneration depended on the animal species. Primates are genetically closer to humans than non-primates; therefore, the use of primates for the study of meniscus regeneration is of value. Cynomolgus macaques have been widely used in biomedical research especially in pharmacology because the macaques metabolism of drugs is close to that of humans. 20 Cynomolgus macaques stand and walk mainly on their hind legs. From this point, it will be more difficult to regenerate the meniscus in cynomolgus macaques than in other non-primate animals. We used cynomolgus macaques from 12 to 13 years of age. Cynomolgus macaques attain sexual maturity between 3 and 4 years old. The longevity of wild cynomolgus macaques is approximately 15 years. This means that cynomolgus macaques at 12 and 13 years old were considered aged. 21 In this study, we used aggregates of synovial MSCs instead of suspension of synovial MSCs for transplantation. We previously reported that transplantation of aggregates of synovial MSCs regenerated the meniscus more effectively than intraarticular injection of synovial MSCs when the number used was the same. 11 For that, There are three possible advantages of aggregates in this scenario. Firstly, aggregation of synovial MSCs increased chondrogenic potentials. 22 Secondly, the number of synovial MSCs attached to the meniscal defect was higher using aggregates of synovial MSCs than when using suspension of synovial MSCs. 11 Thirdly, synovial MSCs survived longer in the knee joint when aggregates of allogenic synovial MSCs were transplanted than when suspension of allogenic synovial MSCs were transplanted. 11 After meniscectomy, aggregates of synovial MSCs remained within the defect by gravity and surface tension. We did not use any scaffold or glue though the primates were allowed to walk freely without immobilization after waking up from anesthesia. According to our previous studies, MSCs transplanted as aggregates existed in the regenerated meniscus focally and partially in rats. 11 We have also observed cartilage regeneration where aggregates of synovial MSCs could rapidly adhere into osteochondral defects by gravity and surface tension, and remain without major losses, in rabbits. 22 The use of aggregates was practical and convenient for the transplantation of MSCs without the need for fixation by glue or scaffold patch. Autologous synovial MSCs were used for this study. Though more efforts and cost are required to prepare autologous synovial MSCs in comparison with allogenic ones, the use of autologous ones possibly avoids an immunorejection and enables synovial MSCs to survive longer than that of allogeneic ones. For clinical application, autologous synovial MSCs will provide safer and better outcomes than allogeneic ones. Synovial MSCs promoted meniscus regeneration in primates. There are three possible mechanisms to account for the results. Firstly, synovial MSCs directly differentiate into meniscal cells. According to our previous study, LacZþ synovial MSCs could be observed in the regenerated meniscus 12 weeks after transplantation in a rat model. 9 Secondly, MSCs produce a wide variety of cytokines and other trophic factors. Our species-specific microarray analyses showed that synovial MSCs increased expressions of hundreds of genes including PRG4, BMP2, and TSG-6 after transplantation into the knee in a rat osteoarthritis model. 23 Thirdly, synovial MSCs induce synovial tissue to the meniscal defect. This was demonstrated in our previous pig studies and a number of papers have provided support for the importance of this induction of synovial tissue to the meniscal lesion for meniscal healing. 13,24,25 Articular cartilage was better preserved in the MSC group than in the control group in all four primates at 16 weeks. We propose two possible explanations for the results observed. Firstly, even an immature meniscus protects the articular cartilage, because the regenerated meniscus in the MSC group was already better than the control group at 8 weeks. Secondly, aggregates of synovial MSCs produce trophic factors to inhibit progression of articular cartilage degeneration as mentioned above. T1rho mapping was used for evaluation of the meniscus and articular cartilage. T1rho mapping provides objective information of biochemical components such as glycosaminoglycan, collagen, and water in cartilaginous tissues. 26 We recently demonstrated a positive correlation between the T1rho value and histological scores in the torn meniscus of pigs. 19 T1rho mapping might be one of the methods to quantify the quality of the regenerated meniscus and articular cartilage. We would like to mention three limitations of this study. Firstly, the sample number was only three at 8 weeks and only four at 16 weeks because we were not able to acquire additional aged cynomolgus macaques. To cover the deficit of statistical analysis, we showed all macroscopic and histological specimens. Secondly, the type of meniscus defect was uncommon. We removed only the anterior half of the medial meniscus because this procedure was less invasive for primates and a reproducible method. Thirdly, we did not perform a biomechanical test for the regenerated meniscus, and the properties of the regenerated meniscus were not examined. In conclusion, transplantation of aggregates of autologous synovial MSCs promoted meniscus regeneration and delayed progression of degeneration of articular cartilage in aged primates. AUTHORS CONTRIBUTIONS S.K. contributed in conception and design, collection of data, analysis and interpretation of data, and manuscript writing. T.M. contributed in conception and design, interpretation of data, and administrative support. Y.N. contributed in collection of data, inter-

9 1282 KONDO ET AL. pretation of data, and analysis of data. H.K. contributed in interpretation of data. T.W. provided financial support. K.T. contributed in interpretation of data. S.K. contributed in collection of data. H.O. contributed in collection of data. S.S. contributed in providing materials. A.O. contributed in providing materials. M.- M. contributed in interpretation of data. I.S. contributed in conception and design, financial support, manuscript writing, and final approval of manuscript. ACKNOWLEDGMENTS This study was supported by the Highway Program for Realization of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) to I.S. and Grants-in-Aid for Scientific Research by the Japan Society for the Promotion of Science to T.W. (No ). We would like to thank Mr. Tsuyoshi Nagata for providing Ziostation2 software to analyze T1rho mapping, Ms. Miyoko Ojima for her expert histological assistance, and Dr. Benjamin L. 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Acad Radiol 11: SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher s website.