Journal of Orthopaedic Science xxx (2017) 1e7
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Original Article
Centralization of extruded medial meniscus delays cartilage degeneration in rats Nobutake Ozeki a, b, Takeshi Muneta c, Kenici Kawabata c, Hideyuki Koga c, Yusuke Nakagawa a, c, Ryusuke Saito c, Mio Udo c, Katsuaki Yanagisawa c, Toshiyuki Ohara c, Tomoyuki Mochizuki d, Kunikazu Tsuji e, Tomoyuki Saito b, Ichiro Sekiya a, * a
Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Japan Department of Orthopaedic Surgery, Yokohama City University, Japan c Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University, Japan d Department of Joint Reconstruction, Graduate School, Tokyo Medical and Dental University, Japan e Department of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 February 2016 Received in revised form 25 November 2016 Accepted 22 January 2017 Available online xxx
Background: Meniscus extrusion often observed in knee osteoarthritis has a strong correlation with the progression of cartilage degeneration and symptom in the patients. We recently reported a novel procedure “arthroscopic centralization” in which the capsule was sutured to the edge of the tibial plateau to reduce meniscus extrusion in the human knee. However, there is no animal model to study the efficacy of this procedure. The purposes of this study were [1] to establish a model of centralization for the extruded medial meniscus in a rat model; and [2] to investigate the chondroprotective effect of this procedure. Methods: Medial meniscus extrusion was induced by the release of the anterior synovial capsule and the transection of the meniscotibial ligament. Centralization was performed by the pulled-out suture technique. Alternatively, control rats had only the medial meniscus extrusion surgery. Medial meniscus extrusion was evaluated by micro-CT and macroscopic findings. Cartilage degeneration of the medial tibial plateau was evaluated macroscopically and histologically. Results: By micro-CT analysis, the medial meniscus extrusion was significantly improved in the centralization group in comparison to the extrusion group throughout the study. Both macroscopically and histologically, the cartilage lesion of the medial tibial plateau was prevented in the centralization group but was apparent in the control group. Conclusions: We developed medial meniscus extrusion in a rat model, and centralization of the extruded medial meniscus by the pull-out suture technique improved the medial meniscus extrusion and delayed cartilage degeneration, though the effect was limited. Centralization is a promising treatment to prevent the progression of osteoarthritis. © 2017 The Authors. Published by Elsevier B.V. on behalf of The Japanese Orthopaedic Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).
1. Introduction Osteoarthritis (OA) is the most prevalent musculoskeletal disease worldwide, causing disabling pain, chronic disability, and reduced quality of life [1]. It is also important to prevent the
* Corresponding author. Center for Stem Cell and Regenerative Medicine, Department of Applied Regenerative Medicine, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. Fax: þ81 3 5803 0192. E-mail address: [email protected] (I. Sekiya).
progression of OA due to the high cost of treatment [2]. There is an emerging interest in focusing on early OA. MRI and ultrasound studies have revealed that the early changes of the knee joint included bone marrow lesion, synovitis, meniscus injury, and meniscus extrusion [3,4]. 3D-MRI enables quantitative analysis of these meniscus pathologies [5,6]. Meniscus extrusion is observed in meniscus injury including root tear [7,8], after partial meniscectomy [9], after meniscal transplantation [10], and most commonly in knee OA [11,12]. There is a strong correlation between the extruded meniscus and the symptom of the patient or joint space narrowing [5,6]. Meniscus
http://dx.doi.org/10.1016/j.jos.2017.01.024 0949-2658/© 2017 The Authors. Published by Elsevier B.V. on behalf of The Japanese Orthopaedic Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Ozeki N, et al., Centralization of extruded medial meniscus delays cartilage degeneration in rats, Journal of Orthopaedic Science (2017), http://dx.doi.org/10.1016/j.jos.2017.01.024
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extrusion is one of the strongest predictors for the progression of OA [13,14], because it decreases meniscal coverage of the tibial plateau, which causes poor distribution of load bearing in the joint. To prevent the progression of OA, intervention against meniscus extrusion would be one option. However, no treatment has yet been reported. This may be due to the lack of treatment options for improving this meniscus pathology. On the other hand, various techniques have been developed for the repair of the meniscus injuries through the development of meniscus repair devices [15]. In human cases, we recently introduced a novel procedure of arthroscopic centralization for an extruded meniscus using suture anchors to stabilize the meniscus on the edge of the tibial plateau [16]. At 3 months postoperative, the extrusion of the meniscus was improved in the MRI findings, and symptoms such as pain and swelling were improved. However, no animal model of centralization for the extruded meniscus is currently available. Therefore, the long-term effects of this procedure have not been studied. The aims of this study were (1) to establish a model of centralization for the extruded medial meniscus in rats, and (2) to investigate the effectiveness of this procedure. 2. Methods 2.1. Animals A total of 52 male wild type Lewis rats at 10e12 weeks old (Charles River Laboratories Japan, Kanagawa, Japan) were used for the experiments. All animal care and experimentation were conducted in accordance with the institutional guidelines of the Animal Committee of Tokyo Medical and Dental University. 2.2. Surgery For anesthesia, isoflurane inhalation was used for anesthesia induction, and intraperitoneal injection of tribromoethanol was used to maintain the sedation (200 mg/kg). All surgeries were performed on the left knee joints. After a straight skin incision, the patellar tendon was dislocated laterally. To induce the extrusion of the medial meniscus, the medial meniscotibial ligament was first transected at the attachment site of tibia using a scalpel [17] and then the anterior synovial capsule, situated between the medial meniscus and the tibial plateau, was transected (Fig. 1Ai). Destabilization was confirmed by distracting the medial meniscus medially with forceps, and extrusion of the medial meniscus was confirmed (Fig. 1Aii). The medial meniscus was then centralized in the experimental group (Fig. 1Aiii). For the centralization of the medial meniscus, a needle with 50 suture nylon (BEAR Medic Corporation, Ibaraki, Japan) was inserted through the peripheral margin of the medial meniscus (Fig. 1Bi). Then the needle with suture nylon was directly passed through the edge of the medial tibial plateau and pulled out to the anterior portion of the proximal tibia through the bone tissue (Fig. 1Bii). After the two sutures were passaged, a knot secured the sutures with the medial meniscus in the original position (Fig. 1Biii). The rats were allowed to walk freely in their cages after the surgery, and their knees were evaluated at 1 day, and 1, 2, 4, and 8 weeks post-surgery (Fig. 1C, N ¼ 5, each group). As a control, the same number of rats received surgery only of medial meniscus extrusion and was evaluated at the same time points post-surgery. Non-surgical, 20 week old rats (n ¼ 2) were used as an additional control, which corresponded to the rat age at the 8 week postoperative time point.
2.3. Micro-CT analyses After the rats were euthanized by CO2 inhalation, whole knee joints were evaluated by micro-CT (Scam Xmate-E090, Comscantechno Co., Kanagawa, Japan). For settings, tube voltage was 70 kV, tube current was 40 mA, and tube power was 2.8 W. Analyses were conducted by using bone structure analysis software (Tri/3D-Bon, RATOC System Engineering Co., Tokyo, Japan). The coronal plane, parallel to the posterior condylar axis of the femoral condyle, and connected to the tip of the growth plate in the axial plane of the tibia, was used for CT analysis (Fig. 2A, white dotted line). It was confirmed that no femorotibial rotation occurred. For the quantitative assessment of the medial meniscus extrusion, Axio Vision Rel software version 4.8 (Carl Zeiss, Oberkochen, Germany) was used. For the quantitative analysis of the medial meniscus extrusion, “meniscus extrusion ratio” and “meniscus coverage ratio” were defined (Fig. 2B). 2.4. Macroscopic observation After the micro-CT analysis, macroscopic features of the medial meniscus on the tibial plateau were evaluated using an Olympus MVX10 macroview microscope (Olympus, Tokyo, Japan), on a dedicated medical photography platform. Then, the medial meniscus was removed and the tibial plateau was stained with India ink for the evaluation of the cartilage degeneration. 2.5. Histological examination After the macroscopic analyses, the medial tibial plateau was further evaluated for the histology. Specimens were fixed in 4% paraformaldehyde for 7 days, decalcified in 20% EDTA solution for 21 days, and embedded in paraffin wax. 5 mm sections were stained with safranin-o and fast green. Histological sections were visualized using an Olympus BX53 microscope (Olympus, Tokyo, Japan). Cartilage degeneration at the center of the medial tibial plateau, which included the most affected lesion, was evaluated using the Mankin score, on a scale of 0e14 points [18], in which a lower score indicates less degeneration. 2.6. Statistical analysis The StatView 5.0 program (SAS Institute, Cary, NC) was used for statistical analyses. Quantitative assessment of the micro-CT analysis and Mankin score were analyzed using the Mann-Whiteny U test. P values less than 0.05 were considered to be statistically significant. 3. Results 3.1. Centralization for the extruded medial meniscus improved the meniscus coverage on the tibial cartilage Micro-CT imaging demonstrated that the medial meniscus was completely displaced out of the medial tibial plateau at 1 day postsurgery in the extrusion group (Fig. 2C), and displacement did not spontaneously improve throughout the 8 week analysis period. In the centralization group, the mid body of the medial meniscus stayed on the medial tibial plateau and the effect was still confirmed at 8 weeks, although moderate extrusion was observed at 1 day post-surgery and thereafter. “Meniscus extrusion ratio” was significantly lower and “meniscus coverage ratio” was significantly higher in the centralization group than in the extrusion group throughout the study (Fig. 2D). Macroscopically, the anterior portion of the medial
Please cite this article in press as: Ozeki N, et al., Centralization of extruded medial meniscus delays cartilage degeneration in rats, Journal of Orthopaedic Science (2017), http://dx.doi.org/10.1016/j.jos.2017.01.024
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Fig. 1. Schema for the experiments. A, Procedure of the centralization for medial meniscus extrusion. (i) Meniscotibial ligament is resected and the medial meniscus is destabilized. Red dots indicate the transection line of the meniscotibial ligament and capsule. (ii) Medial meniscus is extruded. (iii) Medial meniscus is centralized and stabilized using a pulledout suture method. As a control, medial meniscus is left extruded. Black dots indicate the route of the suture passage through the bone. MM; medial meniscus, LM; lateral meniscus, MCL; medial collateral ligament. B, Image of the centralization. (i) Suture is passed through the bone. Arrow indicates the entry point and arrowhead indicates an exit point of the suture. (ii) Two sutures are passed through 2 bone tunnels. (iii) The suture is knotted at the anterior portion of the tibia. An arrowhead indicates the secured knot. C, Evaluation schedule.
meniscus was medially displaced at day 1 in the extrusion-only control group (Fig. 3A). The exposed cartilage, observed at day 1, was gradually covered with the synovial tissue at 1 and 8 weeks. In the centralization group, the displacement of the medial meniscus was slight and the limited exposed cartilage, observed at day 1, was also gradually covered with synovial tissue.
apparent at 4 weeks but did not progress. The Mankin score increased with time in both groups. However, the score was significantly lower at 4 and 8 weeks in the centralization group than in the control group (Fig. 4B).
3.2. Centralization for the extruded medial meniscus delayed cartilage degeneration
Meniscus extrusion is one of the risk factors for progression of OA, and is correlated with symptoms in OA patients. However, no treatment had been attempted for the reduction of this pathology. We recently reported the arthroscopic technique of the centralization for the extruded meniscus for the first time in humans [16], and obtained good results with a short-term follow-up [19]. However, there is no evidence that confirms that the centralization of the extruded meniscus produces a delay in the progression of cartilage degeneration, which directed us to establish the evidence regarding the effectiveness of this procedure. In this study, we proposed a novel method of extrusion of the medial meniscus model to induce OA of the knee in rats. One of the
Macroscopic observation of the medial tibial plateau showed cartilage erosion at 8 weeks in the extrusion group. The cartilage erosion was observed at the center of the medial tibial cartilage rather than at the anterior site of the medial tibial cartilage. Contrarily, cartilage erosion was minimal at 8 weeks in the centralization group (Fig. 3B). Histologically, in the extrusion group, the cartilage degradation became obvious at 2 weeks and progressed throughout the study (Fig. 4A). In the centralization group, cartilage degradation became
4. Discussion
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Fig. 2. Micro-CT analysis for the medial meniscus. A, Index line for the coronal section in the femur and the tibia. The coronal plane, parallel to the posterior condylar axis of the femoral condyle, and connected to the tip of the growth plate in the axial plane of the tibia, was used for CT analysis. B, Definition of “meniscus extrusion ratio” and “meniscus coverage ratio.” Yellow dotted line indicates the line vertically drawn from the medial edge of femoral condyle. C, Coronal view of the knee joint. The index line is shown for the centralized group and the extrusion group (control). D, Meniscus extrusion ratio; quantitation of the extruded meniscus. Bars show the mean ± SD (n ¼ 5 per group). E, Meniscus covering ratio; quantitation of the extruded meniscus. Bars show the mean ± SD (n ¼ 5 per group). * ¼ P < 0.01, * ¼ P < 0.05, by ManneWhitney U test.
popular models that leads to OA in rodents is a destabilization of the medial meniscus (DMM), which is caused by transection of the meniscotibial ligament [17,20]. The DMM model was established for the experiments to evaluate the effect of new drugs [21], and not designed to reduce the destabilized meniscus. Our extrusion of the medial meniscus model is different from the DMM model in that the anterior synovial capsule was released in addition to the transection of meniscotibial ligament. We confirmed the complete dislocation of the anterior part of the medial meniscus out of the medial tibial plateau. Although the invasiveness of the 2 models is similar, our extrusion of the medial meniscus model reliably produces an extrusion of the anterior part of the medial meniscus.
Therefore, this technique is suitable for the evaluation of novel treatment for meniscus pathologies. An impaired meniscus causes OA of the knee and this condition is mimicked by an extrusion of the medial meniscus. Previously, abundant experiments were tried for meniscus repair [22], meniscus transplantation [23], or meniscus regeneration [24]. However, there are no reports investigating the effect of centralization, which reduces and stabilizes the extruded meniscus in the original position at the edge of the tibial plateau. The lack of meniscus centralization in prior studies may be due to both the oversight of the need and the lack of method to accomplish the reduction of the extruded meniscus. In this study, we focused on
Please cite this article in press as: Ozeki N, et al., Centralization of extruded medial meniscus delays cartilage degeneration in rats, Journal of Orthopaedic Science (2017), http://dx.doi.org/10.1016/j.jos.2017.01.024
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Fig. 3. Representative macroscopic features. A, Medial meniscus on the medial tibial plateau. Red dots indicate the center line of the tibial plateau. Arrowhead indicates the attachment of the anterior portion of the medial meniscus. An arrow indicates the exposed cartilage due to the displaced medial meniscus. A; anterior side, P; posterior side. B, Medial tibial plateau stained with India ink after medial and lateral menisci were removed. Arrow indicates the cartilage lesion.
the treatment of the extruded medial meniscus to produce an animal model for future studies on the effect of the medial meniscus centralization on the progression of OA. We transected both the anterior insertional ligament and anterior synovium to induce extrusion of the medial meniscus. Repairing these tissues leads to the repositioning of the extruded medial meniscus. In fact, in future studies it would also be reasonable to repair the insertion of the meniscus for meniscus root tear, which also causes meniscus extrusion [25]. However, since the aim of this study was to improve the meniscus extrusion that occurred in common OA without meniscus root tear, we aimed to centralize the extruded meniscus without repairing these tissues. To achieve the centralization for the extruded medial meniscus in rats, we adopted a pulled-out suture technique. This technique is a different procedure from the suture anchor technique in humans that we previously reported [16,19] due to the lack of suitable anchors for small animals. However, the concept of the centralization is the same. In this study, suture nylon was passed through the peripheral margin of the medial meniscus and the edge of the medial tibial plateau, resulting in the extruded medial meniscus being secured on the tibial plateau. Although this procedure is technically demanding in rats and the entry point of the suture is critical, it produced a reproducible improvement of medial meniscus displacement. Therefore, this procedure is a reasonable
animal model for examining the effect of medial meniscus centralization on the progression of OA. One day after the extruded medial meniscus was centralized toward the original position, the medial meniscus moved medially again to a certain extent at day 1. We propose 2 reasons for this. Firstly, we could not maintain the medial meniscus at the anatomical position even just after the surgery due to a low strength of 5-0 nylon. Secondly, the medial meniscus was extruded again 24 h after the surgery, possibly because rats walked around after the anesthetic wore off. In this study, we demonstrated that the pull-out suture technique improved the medial meniscus extrusion but the effect was limited. MRI is usually used in clinical situations for the assessment of meniscus extrusion. Several reports in rats attempted to assess the cartilage or meniscus by MRI, but it is not useful to evaluate these tissues in small animals because of the poor resolution of the images [26]. Due to the limited resolution of MRI, we utilized microCT to analyze the rat medial meniscus. The innate calcification of the rat meniscus enables us to confirm the position of the medial meniscus body in the coronal section of micro-CT [27]. Micro-CT cannot evaluate the quality of the meniscus, but the resolution is sufficient to analyze the extent of extrusion. Once the medial meniscus was extruded, the displacement was not improved without future intervention, replicating the result of
Please cite this article in press as: Ozeki N, et al., Centralization of extruded medial meniscus delays cartilage degeneration in rats, Journal of Orthopaedic Science (2017), http://dx.doi.org/10.1016/j.jos.2017.01.024
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Fig. 4. Histological analysis of the medial tibial cartilage. A, Sagittal sections of the medial tibial cartilage stained with safranin-o. The most affected lesion was magnified and high magnification of the square in the upper panel is shown in the lower panel. A; anterior side, P; posterior side. B, Mankin score. Bars show the mean ± SD (n ¼ 5 rats per group). * ¼ P < 0.05 by ManneWhitney U test.
clinical situations [4]. Conversely, centralization of the extruded medial meniscus produced the desired meniscal coverage of the medial tibial plateau, and the effect remained throughout the 8 week experimental period. Most importantly, centralization for the extruded medial meniscus significantly delayed cartilage degeneration, which indicates that the meniscal coverage of the medial tibial plateau is an important factor for the protection of the cartilage. MRI studies in humans that showed the correlation of meniscus extrusion and the OA progression indicated the importance of the meniscal coverage [6,28], which was replicated in this experimental model. Our report of meniscus centralization in humans showed an improvement of knee symptom, but due to the short follow-up period, the long-term chondroprotective effect was unclear [16]. The current study is the first to show that the centralization technique for an extruded meniscus can delay cartilage degeneration. Though the anterior medial meniscus was extruded, the cartilage degeneration occurred at the center of the medial tibial cartilage rather than the anterior part of the medial tibial cartilage. In other studies of ours, we investigated the influence of the anterior medial meniscus extrusion on distribution of the loading force in the knee and the load was concentrated mainly to the center of the medial tibial cartilage in pig models (data not shown). We also found that cartilage degeneration occurred at the center of the medial tibial cartilage rather than the anterior part of the medial tibial cartilage in rats [24,29,30].
These results indicate that dysfunction of the anterior medial meniscus leads to degenerate cartilage at the center of the medial tibial cartilage rather than at the anterior part of the medial tibial cartilage. We propose several limitations in this study. Though the posterior body of the medial meniscus could be more involved in the pathology relevant to the extrusion than the anterior body in clinical situations, we destabilized the anterior medial meniscus because destabilization of the posterior medial meniscus was highly invasive and complicated. In our current model, the medial collateral ligament could be preserved, and we could complete this model with low invasiveness, with relative ease, and with high reproducibility. Though our model mimics limited pathological conditions of meniscal lesions, it was useful to investigate the effectiveness of an unestablished treatment to improve meniscal extrusion. In the next step, we would like to prove our technique in a posterior medial meniscus extrusion model using larger animals such as minipigs. In regard to other limitation, this study lacked the biomechanical analysis for the centralized medial meniscus. Therefore, we cannot confirm that the centralized medial meniscus has the same biomechanical properties as the normal medial meniscus. However, the chondroprotective result of centralization after medial meniscus extrusion suggests that the biomechanical properties of the centralized medial meniscus functioned closer to the native rat medial meniscus than the extruded medial meniscus.
Please cite this article in press as: Ozeki N, et al., Centralization of extruded medial meniscus delays cartilage degeneration in rats, Journal of Orthopaedic Science (2017), http://dx.doi.org/10.1016/j.jos.2017.01.024
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We performed the centralization technique in the acute animal model of the medial meniscus extrusion, which does not replicate clinical cases involving the gradual progress of medial meniscus extrusion. Therefore, in the next study, we would like to evaluate the effectiveness of this procedure in rats where extrusion of the medial meniscus occurs several weeks before the centralization procedure. The animal model we used was insufficient to obtain clinical relevance. Rats are small animals whose knee joints are obviously different from humans, therefore, rats cannot provide fully clinically relevant models especially with regards to studies of the meniscus. Looking forward, large animal models are required to investigate the effect of the centralization of extruded menisci. In conclusion, we developed an extrusion of the medial meniscus model in rats to induce OA of the knee, and centralization for the extruded meniscus improved the meniscus coverage of the tibial cartilage, which delayed cartilage degeneration. Author contributions NO: Conception and design. Collection, analysis, and interpretation of data. TM: Conception and design. KK; Collection of data. HK: Conception and design. YN: Interpretation of data. RS: Collection of data. MU: Collection of data. KY: Collection of data. TO: Collection of data. TM: Interpretation of data. KT: Interpretation of data. TS: Interpretation of data. IS: Conception and design, final approval of the article. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We would like to thank Ms. Miyoko Ojima for her expert help with the study. This study was supported by “the Highway Program for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology” and “a grant-inaid for Research on Regenerative Medicine for Clinical Application from the Ministry of Health, Labor and Welfare”. References [1] Chevalier X, Eymard F, Richette P. Biologic agents in osteoarthritis: hopes and disappointments. Nat Rev Rheumatol 2013 Jul;9(7):400e10. [2] Zhang W, Nuki G, Moskowitz RW, Abramson S, Altman RD, Arden NK, BiermaZeinstra S, Brandt KD, Croft P, Doherty M, Dougados M, Hochberg M, Hunter DJ, Kwoh K, Lohmander LS, Tugwell P. OARSI recommendations for the management of hip and knee osteoarthritis: part III: changes in evidence following systematic cumulative update of research published through January 2009. Osteoarthr Cartil 2010 Apr;18(4):476e99. [3] Felson DT, Lynch J, Guermazi A, Roemer FW, Niu J, McAlindon T, Nevitt MC. Comparison of BLOKS and WORMS scoring systems part II. Longitudinal assessment of knee MRIs for osteoarthritis and suggested approach based on their performance: data from the Osteoarthritis Initiative. Osteoarthr Cartil 2010 Nov;18(11):1402e7. [4] Kawaguchi K, Enokida M, Otsuki R, Teshima R. Ultrasonographic evaluation of medial radial displacement of the medial meniscus in knee osteoarthritis. Arthritis Rheum 2012 Jan;64(1):173e80. [5] Wenger A, Englund M, Wirth W, Hudelmaier M, Kwoh K, Eckstein F, Investigators OAI. Relationship of 3D meniscal morphology and position with knee pain in subjects with knee osteoarthritis: a pilot study. Eur Radiol 2012 Jan;22(1):211e20. [6] Bloecker K, Guermazi A, Wirth W, Benichou O, Kwoh CK, Hunter DJ, Englund M, Resch H, Eckstein F, Investigators OAI. Tibial coverage, meniscus position, size and damage in knees discordant for joint space narrowing e data from the osteoarthritis initiative. Osteoarthr Cartil 2013 Mar;21(3):419e27. [7] Anderson L, Watts M, Shapter O, Logan M, Risebury M, Duffy D, Myers P. Repair of radial tears and posterior horn detachments of the lateral meniscus: minimum 2-year follow-up. Arthroscopy 2010 Dec;26(12):1625e32.
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Original Article
Centralization of extruded medial meniscus delays cartilage degeneration in rats Nobutake Ozeki a, b, Takeshi Muneta c, Kenici Kawabata c, Hideyuki Koga c, Yusuke Nakagawa a, c, Ryusuke Saito c, Mio Udo c, Katsuaki Yanagisawa c, Toshiyuki Ohara c, Tomoyuki Mochizuki d, Kunikazu Tsuji e, Tomoyuki Saito b, Ichiro Sekiya a, * a
Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Japan Department of Orthopaedic Surgery, Yokohama City University, Japan c Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University, Japan d Department of Joint Reconstruction, Graduate School, Tokyo Medical and Dental University, Japan e Department of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 February 2016 Received in revised form 25 November 2016 Accepted 22 January 2017 Available online xxx
Background: Meniscus extrusion often observed in knee osteoarthritis has a strong correlation with the progression of cartilage degeneration and symptom in the patients. We recently reported a novel procedure “arthroscopic centralization” in which the capsule was sutured to the edge of the tibial plateau to reduce meniscus extrusion in the human knee. However, there is no animal model to study the efficacy of this procedure. The purposes of this study were [1] to establish a model of centralization for the extruded medial meniscus in a rat model; and [2] to investigate the chondroprotective effect of this procedure. Methods: Medial meniscus extrusion was induced by the release of the anterior synovial capsule and the transection of the meniscotibial ligament. Centralization was performed by the pulled-out suture technique. Alternatively, control rats had only the medial meniscus extrusion surgery. Medial meniscus extrusion was evaluated by micro-CT and macroscopic findings. Cartilage degeneration of the medial tibial plateau was evaluated macroscopically and histologically. Results: By micro-CT analysis, the medial meniscus extrusion was significantly improved in the centralization group in comparison to the extrusion group throughout the study. Both macroscopically and histologically, the cartilage lesion of the medial tibial plateau was prevented in the centralization group but was apparent in the control group. Conclusions: We developed medial meniscus extrusion in a rat model, and centralization of the extruded medial meniscus by the pull-out suture technique improved the medial meniscus extrusion and delayed cartilage degeneration, though the effect was limited. Centralization is a promising treatment to prevent the progression of osteoarthritis. © 2017 The Authors. Published by Elsevier B.V. on behalf of The Japanese Orthopaedic Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).
1. Introduction Osteoarthritis (OA) is the most prevalent musculoskeletal disease worldwide, causing disabling pain, chronic disability, and reduced quality of life [1]. It is also important to prevent the
* Corresponding author. Center for Stem Cell and Regenerative Medicine, Department of Applied Regenerative Medicine, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. Fax: þ81 3 5803 0192. E-mail address: [email protected] (I. Sekiya).
progression of OA due to the high cost of treatment [2]. There is an emerging interest in focusing on early OA. MRI and ultrasound studies have revealed that the early changes of the knee joint included bone marrow lesion, synovitis, meniscus injury, and meniscus extrusion [3,4]. 3D-MRI enables quantitative analysis of these meniscus pathologies [5,6]. Meniscus extrusion is observed in meniscus injury including root tear [7,8], after partial meniscectomy [9], after meniscal transplantation [10], and most commonly in knee OA [11,12]. There is a strong correlation between the extruded meniscus and the symptom of the patient or joint space narrowing [5,6]. Meniscus
http://dx.doi.org/10.1016/j.jos.2017.01.024 0949-2658/© 2017 The Authors. Published by Elsevier B.V. on behalf of The Japanese Orthopaedic Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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extrusion is one of the strongest predictors for the progression of OA [13,14], because it decreases meniscal coverage of the tibial plateau, which causes poor distribution of load bearing in the joint. To prevent the progression of OA, intervention against meniscus extrusion would be one option. However, no treatment has yet been reported. This may be due to the lack of treatment options for improving this meniscus pathology. On the other hand, various techniques have been developed for the repair of the meniscus injuries through the development of meniscus repair devices [15]. In human cases, we recently introduced a novel procedure of arthroscopic centralization for an extruded meniscus using suture anchors to stabilize the meniscus on the edge of the tibial plateau [16]. At 3 months postoperative, the extrusion of the meniscus was improved in the MRI findings, and symptoms such as pain and swelling were improved. However, no animal model of centralization for the extruded meniscus is currently available. Therefore, the long-term effects of this procedure have not been studied. The aims of this study were (1) to establish a model of centralization for the extruded medial meniscus in rats, and (2) to investigate the effectiveness of this procedure. 2. Methods 2.1. Animals A total of 52 male wild type Lewis rats at 10e12 weeks old (Charles River Laboratories Japan, Kanagawa, Japan) were used for the experiments. All animal care and experimentation were conducted in accordance with the institutional guidelines of the Animal Committee of Tokyo Medical and Dental University. 2.2. Surgery For anesthesia, isoflurane inhalation was used for anesthesia induction, and intraperitoneal injection of tribromoethanol was used to maintain the sedation (200 mg/kg). All surgeries were performed on the left knee joints. After a straight skin incision, the patellar tendon was dislocated laterally. To induce the extrusion of the medial meniscus, the medial meniscotibial ligament was first transected at the attachment site of tibia using a scalpel [17] and then the anterior synovial capsule, situated between the medial meniscus and the tibial plateau, was transected (Fig. 1Ai). Destabilization was confirmed by distracting the medial meniscus medially with forceps, and extrusion of the medial meniscus was confirmed (Fig. 1Aii). The medial meniscus was then centralized in the experimental group (Fig. 1Aiii). For the centralization of the medial meniscus, a needle with 50 suture nylon (BEAR Medic Corporation, Ibaraki, Japan) was inserted through the peripheral margin of the medial meniscus (Fig. 1Bi). Then the needle with suture nylon was directly passed through the edge of the medial tibial plateau and pulled out to the anterior portion of the proximal tibia through the bone tissue (Fig. 1Bii). After the two sutures were passaged, a knot secured the sutures with the medial meniscus in the original position (Fig. 1Biii). The rats were allowed to walk freely in their cages after the surgery, and their knees were evaluated at 1 day, and 1, 2, 4, and 8 weeks post-surgery (Fig. 1C, N ¼ 5, each group). As a control, the same number of rats received surgery only of medial meniscus extrusion and was evaluated at the same time points post-surgery. Non-surgical, 20 week old rats (n ¼ 2) were used as an additional control, which corresponded to the rat age at the 8 week postoperative time point.
2.3. Micro-CT analyses After the rats were euthanized by CO2 inhalation, whole knee joints were evaluated by micro-CT (Scam Xmate-E090, Comscantechno Co., Kanagawa, Japan). For settings, tube voltage was 70 kV, tube current was 40 mA, and tube power was 2.8 W. Analyses were conducted by using bone structure analysis software (Tri/3D-Bon, RATOC System Engineering Co., Tokyo, Japan). The coronal plane, parallel to the posterior condylar axis of the femoral condyle, and connected to the tip of the growth plate in the axial plane of the tibia, was used for CT analysis (Fig. 2A, white dotted line). It was confirmed that no femorotibial rotation occurred. For the quantitative assessment of the medial meniscus extrusion, Axio Vision Rel software version 4.8 (Carl Zeiss, Oberkochen, Germany) was used. For the quantitative analysis of the medial meniscus extrusion, “meniscus extrusion ratio” and “meniscus coverage ratio” were defined (Fig. 2B). 2.4. Macroscopic observation After the micro-CT analysis, macroscopic features of the medial meniscus on the tibial plateau were evaluated using an Olympus MVX10 macroview microscope (Olympus, Tokyo, Japan), on a dedicated medical photography platform. Then, the medial meniscus was removed and the tibial plateau was stained with India ink for the evaluation of the cartilage degeneration. 2.5. Histological examination After the macroscopic analyses, the medial tibial plateau was further evaluated for the histology. Specimens were fixed in 4% paraformaldehyde for 7 days, decalcified in 20% EDTA solution for 21 days, and embedded in paraffin wax. 5 mm sections were stained with safranin-o and fast green. Histological sections were visualized using an Olympus BX53 microscope (Olympus, Tokyo, Japan). Cartilage degeneration at the center of the medial tibial plateau, which included the most affected lesion, was evaluated using the Mankin score, on a scale of 0e14 points [18], in which a lower score indicates less degeneration. 2.6. Statistical analysis The StatView 5.0 program (SAS Institute, Cary, NC) was used for statistical analyses. Quantitative assessment of the micro-CT analysis and Mankin score were analyzed using the Mann-Whiteny U test. P values less than 0.05 were considered to be statistically significant. 3. Results 3.1. Centralization for the extruded medial meniscus improved the meniscus coverage on the tibial cartilage Micro-CT imaging demonstrated that the medial meniscus was completely displaced out of the medial tibial plateau at 1 day postsurgery in the extrusion group (Fig. 2C), and displacement did not spontaneously improve throughout the 8 week analysis period. In the centralization group, the mid body of the medial meniscus stayed on the medial tibial plateau and the effect was still confirmed at 8 weeks, although moderate extrusion was observed at 1 day post-surgery and thereafter. “Meniscus extrusion ratio” was significantly lower and “meniscus coverage ratio” was significantly higher in the centralization group than in the extrusion group throughout the study (Fig. 2D). Macroscopically, the anterior portion of the medial
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Fig. 1. Schema for the experiments. A, Procedure of the centralization for medial meniscus extrusion. (i) Meniscotibial ligament is resected and the medial meniscus is destabilized. Red dots indicate the transection line of the meniscotibial ligament and capsule. (ii) Medial meniscus is extruded. (iii) Medial meniscus is centralized and stabilized using a pulledout suture method. As a control, medial meniscus is left extruded. Black dots indicate the route of the suture passage through the bone. MM; medial meniscus, LM; lateral meniscus, MCL; medial collateral ligament. B, Image of the centralization. (i) Suture is passed through the bone. Arrow indicates the entry point and arrowhead indicates an exit point of the suture. (ii) Two sutures are passed through 2 bone tunnels. (iii) The suture is knotted at the anterior portion of the tibia. An arrowhead indicates the secured knot. C, Evaluation schedule.
meniscus was medially displaced at day 1 in the extrusion-only control group (Fig. 3A). The exposed cartilage, observed at day 1, was gradually covered with the synovial tissue at 1 and 8 weeks. In the centralization group, the displacement of the medial meniscus was slight and the limited exposed cartilage, observed at day 1, was also gradually covered with synovial tissue.
apparent at 4 weeks but did not progress. The Mankin score increased with time in both groups. However, the score was significantly lower at 4 and 8 weeks in the centralization group than in the control group (Fig. 4B).
3.2. Centralization for the extruded medial meniscus delayed cartilage degeneration
Meniscus extrusion is one of the risk factors for progression of OA, and is correlated with symptoms in OA patients. However, no treatment had been attempted for the reduction of this pathology. We recently reported the arthroscopic technique of the centralization for the extruded meniscus for the first time in humans [16], and obtained good results with a short-term follow-up [19]. However, there is no evidence that confirms that the centralization of the extruded meniscus produces a delay in the progression of cartilage degeneration, which directed us to establish the evidence regarding the effectiveness of this procedure. In this study, we proposed a novel method of extrusion of the medial meniscus model to induce OA of the knee in rats. One of the
Macroscopic observation of the medial tibial plateau showed cartilage erosion at 8 weeks in the extrusion group. The cartilage erosion was observed at the center of the medial tibial cartilage rather than at the anterior site of the medial tibial cartilage. Contrarily, cartilage erosion was minimal at 8 weeks in the centralization group (Fig. 3B). Histologically, in the extrusion group, the cartilage degradation became obvious at 2 weeks and progressed throughout the study (Fig. 4A). In the centralization group, cartilage degradation became
4. Discussion
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Fig. 2. Micro-CT analysis for the medial meniscus. A, Index line for the coronal section in the femur and the tibia. The coronal plane, parallel to the posterior condylar axis of the femoral condyle, and connected to the tip of the growth plate in the axial plane of the tibia, was used for CT analysis. B, Definition of “meniscus extrusion ratio” and “meniscus coverage ratio.” Yellow dotted line indicates the line vertically drawn from the medial edge of femoral condyle. C, Coronal view of the knee joint. The index line is shown for the centralized group and the extrusion group (control). D, Meniscus extrusion ratio; quantitation of the extruded meniscus. Bars show the mean ± SD (n ¼ 5 per group). E, Meniscus covering ratio; quantitation of the extruded meniscus. Bars show the mean ± SD (n ¼ 5 per group). * ¼ P < 0.01, * ¼ P < 0.05, by ManneWhitney U test.
popular models that leads to OA in rodents is a destabilization of the medial meniscus (DMM), which is caused by transection of the meniscotibial ligament [17,20]. The DMM model was established for the experiments to evaluate the effect of new drugs [21], and not designed to reduce the destabilized meniscus. Our extrusion of the medial meniscus model is different from the DMM model in that the anterior synovial capsule was released in addition to the transection of meniscotibial ligament. We confirmed the complete dislocation of the anterior part of the medial meniscus out of the medial tibial plateau. Although the invasiveness of the 2 models is similar, our extrusion of the medial meniscus model reliably produces an extrusion of the anterior part of the medial meniscus.
Therefore, this technique is suitable for the evaluation of novel treatment for meniscus pathologies. An impaired meniscus causes OA of the knee and this condition is mimicked by an extrusion of the medial meniscus. Previously, abundant experiments were tried for meniscus repair [22], meniscus transplantation [23], or meniscus regeneration [24]. However, there are no reports investigating the effect of centralization, which reduces and stabilizes the extruded meniscus in the original position at the edge of the tibial plateau. The lack of meniscus centralization in prior studies may be due to both the oversight of the need and the lack of method to accomplish the reduction of the extruded meniscus. In this study, we focused on
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Fig. 3. Representative macroscopic features. A, Medial meniscus on the medial tibial plateau. Red dots indicate the center line of the tibial plateau. Arrowhead indicates the attachment of the anterior portion of the medial meniscus. An arrow indicates the exposed cartilage due to the displaced medial meniscus. A; anterior side, P; posterior side. B, Medial tibial plateau stained with India ink after medial and lateral menisci were removed. Arrow indicates the cartilage lesion.
the treatment of the extruded medial meniscus to produce an animal model for future studies on the effect of the medial meniscus centralization on the progression of OA. We transected both the anterior insertional ligament and anterior synovium to induce extrusion of the medial meniscus. Repairing these tissues leads to the repositioning of the extruded medial meniscus. In fact, in future studies it would also be reasonable to repair the insertion of the meniscus for meniscus root tear, which also causes meniscus extrusion [25]. However, since the aim of this study was to improve the meniscus extrusion that occurred in common OA without meniscus root tear, we aimed to centralize the extruded meniscus without repairing these tissues. To achieve the centralization for the extruded medial meniscus in rats, we adopted a pulled-out suture technique. This technique is a different procedure from the suture anchor technique in humans that we previously reported [16,19] due to the lack of suitable anchors for small animals. However, the concept of the centralization is the same. In this study, suture nylon was passed through the peripheral margin of the medial meniscus and the edge of the medial tibial plateau, resulting in the extruded medial meniscus being secured on the tibial plateau. Although this procedure is technically demanding in rats and the entry point of the suture is critical, it produced a reproducible improvement of medial meniscus displacement. Therefore, this procedure is a reasonable
animal model for examining the effect of medial meniscus centralization on the progression of OA. One day after the extruded medial meniscus was centralized toward the original position, the medial meniscus moved medially again to a certain extent at day 1. We propose 2 reasons for this. Firstly, we could not maintain the medial meniscus at the anatomical position even just after the surgery due to a low strength of 5-0 nylon. Secondly, the medial meniscus was extruded again 24 h after the surgery, possibly because rats walked around after the anesthetic wore off. In this study, we demonstrated that the pull-out suture technique improved the medial meniscus extrusion but the effect was limited. MRI is usually used in clinical situations for the assessment of meniscus extrusion. Several reports in rats attempted to assess the cartilage or meniscus by MRI, but it is not useful to evaluate these tissues in small animals because of the poor resolution of the images [26]. Due to the limited resolution of MRI, we utilized microCT to analyze the rat medial meniscus. The innate calcification of the rat meniscus enables us to confirm the position of the medial meniscus body in the coronal section of micro-CT [27]. Micro-CT cannot evaluate the quality of the meniscus, but the resolution is sufficient to analyze the extent of extrusion. Once the medial meniscus was extruded, the displacement was not improved without future intervention, replicating the result of
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Fig. 4. Histological analysis of the medial tibial cartilage. A, Sagittal sections of the medial tibial cartilage stained with safranin-o. The most affected lesion was magnified and high magnification of the square in the upper panel is shown in the lower panel. A; anterior side, P; posterior side. B, Mankin score. Bars show the mean ± SD (n ¼ 5 rats per group). * ¼ P < 0.05 by ManneWhitney U test.
clinical situations [4]. Conversely, centralization of the extruded medial meniscus produced the desired meniscal coverage of the medial tibial plateau, and the effect remained throughout the 8 week experimental period. Most importantly, centralization for the extruded medial meniscus significantly delayed cartilage degeneration, which indicates that the meniscal coverage of the medial tibial plateau is an important factor for the protection of the cartilage. MRI studies in humans that showed the correlation of meniscus extrusion and the OA progression indicated the importance of the meniscal coverage [6,28], which was replicated in this experimental model. Our report of meniscus centralization in humans showed an improvement of knee symptom, but due to the short follow-up period, the long-term chondroprotective effect was unclear [16]. The current study is the first to show that the centralization technique for an extruded meniscus can delay cartilage degeneration. Though the anterior medial meniscus was extruded, the cartilage degeneration occurred at the center of the medial tibial cartilage rather than the anterior part of the medial tibial cartilage. In other studies of ours, we investigated the influence of the anterior medial meniscus extrusion on distribution of the loading force in the knee and the load was concentrated mainly to the center of the medial tibial cartilage in pig models (data not shown). We also found that cartilage degeneration occurred at the center of the medial tibial cartilage rather than the anterior part of the medial tibial cartilage in rats [24,29,30].
These results indicate that dysfunction of the anterior medial meniscus leads to degenerate cartilage at the center of the medial tibial cartilage rather than at the anterior part of the medial tibial cartilage. We propose several limitations in this study. Though the posterior body of the medial meniscus could be more involved in the pathology relevant to the extrusion than the anterior body in clinical situations, we destabilized the anterior medial meniscus because destabilization of the posterior medial meniscus was highly invasive and complicated. In our current model, the medial collateral ligament could be preserved, and we could complete this model with low invasiveness, with relative ease, and with high reproducibility. Though our model mimics limited pathological conditions of meniscal lesions, it was useful to investigate the effectiveness of an unestablished treatment to improve meniscal extrusion. In the next step, we would like to prove our technique in a posterior medial meniscus extrusion model using larger animals such as minipigs. In regard to other limitation, this study lacked the biomechanical analysis for the centralized medial meniscus. Therefore, we cannot confirm that the centralized medial meniscus has the same biomechanical properties as the normal medial meniscus. However, the chondroprotective result of centralization after medial meniscus extrusion suggests that the biomechanical properties of the centralized medial meniscus functioned closer to the native rat medial meniscus than the extruded medial meniscus.
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We performed the centralization technique in the acute animal model of the medial meniscus extrusion, which does not replicate clinical cases involving the gradual progress of medial meniscus extrusion. Therefore, in the next study, we would like to evaluate the effectiveness of this procedure in rats where extrusion of the medial meniscus occurs several weeks before the centralization procedure. The animal model we used was insufficient to obtain clinical relevance. Rats are small animals whose knee joints are obviously different from humans, therefore, rats cannot provide fully clinically relevant models especially with regards to studies of the meniscus. Looking forward, large animal models are required to investigate the effect of the centralization of extruded menisci. In conclusion, we developed an extrusion of the medial meniscus model in rats to induce OA of the knee, and centralization for the extruded meniscus improved the meniscus coverage of the tibial cartilage, which delayed cartilage degeneration. Author contributions NO: Conception and design. Collection, analysis, and interpretation of data. TM: Conception and design. KK; Collection of data. HK: Conception and design. YN: Interpretation of data. RS: Collection of data. MU: Collection of data. KY: Collection of data. TO: Collection of data. TM: Interpretation of data. KT: Interpretation of data. TS: Interpretation of data. IS: Conception and design, final approval of the article. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We would like to thank Ms. Miyoko Ojima for her expert help with the study. This study was supported by “the Highway Program for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology” and “a grant-inaid for Research on Regenerative Medicine for Clinical Application from the Ministry of Health, Labor and Welfare”. References [1] Chevalier X, Eymard F, Richette P. Biologic agents in osteoarthritis: hopes and disappointments. Nat Rev Rheumatol 2013 Jul;9(7):400e10. [2] Zhang W, Nuki G, Moskowitz RW, Abramson S, Altman RD, Arden NK, BiermaZeinstra S, Brandt KD, Croft P, Doherty M, Dougados M, Hochberg M, Hunter DJ, Kwoh K, Lohmander LS, Tugwell P. OARSI recommendations for the management of hip and knee osteoarthritis: part III: changes in evidence following systematic cumulative update of research published through January 2009. Osteoarthr Cartil 2010 Apr;18(4):476e99. [3] Felson DT, Lynch J, Guermazi A, Roemer FW, Niu J, McAlindon T, Nevitt MC. Comparison of BLOKS and WORMS scoring systems part II. Longitudinal assessment of knee MRIs for osteoarthritis and suggested approach based on their performance: data from the Osteoarthritis Initiative. Osteoarthr Cartil 2010 Nov;18(11):1402e7. [4] Kawaguchi K, Enokida M, Otsuki R, Teshima R. Ultrasonographic evaluation of medial radial displacement of the medial meniscus in knee osteoarthritis. Arthritis Rheum 2012 Jan;64(1):173e80. [5] Wenger A, Englund M, Wirth W, Hudelmaier M, Kwoh K, Eckstein F, Investigators OAI. Relationship of 3D meniscal morphology and position with knee pain in subjects with knee osteoarthritis: a pilot study. Eur Radiol 2012 Jan;22(1):211e20. [6] Bloecker K, Guermazi A, Wirth W, Benichou O, Kwoh CK, Hunter DJ, Englund M, Resch H, Eckstein F, Investigators OAI. Tibial coverage, meniscus position, size and damage in knees discordant for joint space narrowing e data from the osteoarthritis initiative. Osteoarthr Cartil 2013 Mar;21(3):419e27. [7] Anderson L, Watts M, Shapter O, Logan M, Risebury M, Duffy D, Myers P. Repair of radial tears and posterior horn detachments of the lateral meniscus: minimum 2-year follow-up. Arthroscopy 2010 Dec;26(12):1625e32.
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Please cite this article in press as: Ozeki N, et al., Centralization of extruded medial meniscus delays cartilage degeneration in rats, Journal of Orthopaedic Science (2017), http://dx.doi.org/10.1016/j.jos.2017.01.024
