Effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of anterior cruciate ligament injury in a rat model

Effects of controlled abnormal joint movement on the molecular biological response in... Background: The anterior cruciate ligament (ACL) is responsible for braking forward movement of the tibia relative to the femur and for tibial rotation. After ACL injury, this braking performance deteriorates, inducing abnormal joint movement. The purpose of this study was to clarify the effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of ACL injury. Methods: Eighty-four mature Wistar male rats were randomly assigned to a controlled abnormal movement (CAM) group, an ACL-transection (ACL-T) group, a sham-operated group, or an intact group. The ACL was completely transected at its midportion in the ACL-T and CAM groups, and a nylon suture was used to control abnormal tibial translation in the CAM group. The sham-operated group underwent skin and joint capsule incisions and tibial drilling without ACL transection. Animals were not restricted activity until sacrifice 1, 3, or 5 days after surgery for histological and gene expression assessments. Acute-phase inflammation requires an important balance between degenerative and biosynthetic processes and is controlled by the activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Both types of gene were analyzed in this study. Results: The ACL-T and CAM groups exhibited cleavage of the ACL at all time points. However, for the CAM group, the gap in the ligament stump was extremely small, and fibroblast proliferation was observed around the stump. Relative to the ACL-T group, the CAM group demonstrated significantly lower expression of MMP-13 mRNA and a lower MMP-13/TIMP-1 ratio on days 1 and 5 in the ACL, the medial meniscus and the lateral meniscus. The expression of TIMP-1 mRNA was not significantly different between the ACL-T and CAM groups. Conclusions: The study results suggested that controlling abnormal movement inhibited the inflammatory reaction in intra-articular tissues after ACL injury. This reaction was down-regulated in intra-articular tissues in the CAM group. Abnormal joint control caused prolonged inflammation and inhibited remodeling during the acute phase of ACL rupture. Keywords: Anterior cruciate ligament, Matrix metalloproteinase-13, Conservative therapy, Acute phase injury * Correspondence: takayanagi-kiyomi@spu.ac.jp Department of Physical Therapy, School of Health and Social Services, Saitama Prefectural University, 820 Sannomiya, Koshigaya-shi, Saitama 343-8540, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 2 of 9 Background unclear. It is thought that controlled abnormal joint move- The anterior cruciate ligament (ACL) plays an important ment is an important factor for spontaneous ACL healing. role in controlling and stabilizing the knee joint; it is the Many previous studies have reported the molecular primary restraint against anterior tibial translation [1]. biological responses in the intra-articular tissues dur- The prevalence of injury to the ACL is quite high, particu- ing the acute phase [28–31]. However, to the best of larly among athletes who perform pivoting activities [2, 3]. the authors’ knowledge, no reports have yet focused on the Furthermore, previous reported that female subjects signifi- effects of abnormal joint movement on intra-articular tis- cantly higher risk of ACL injury than male subjects [4, 5]. sues. The acute-phase inflammatory response plays an im- Treatment after ACL injury often involves reconstructive portant role in the wound healing process, and the balance surgery, which is performed on more than half of ACL in- between the degenerative and biosynthetic arms of this jury patients [6, 7]. process is controlled by the activities of matrix metallopro- ACL deficiency can induce the degeneration of other teinases (MMPs) and tissue inhibitors of metalloproteinases intra-articular tissues (i.e., cartilage and meniscus), which is (TIMPs) [32]. In the present study, we focused on MMPs a risk factor for the development of osteoarthritis [8]. Previ- and TIMPs to determine the molecular biological response ous studies have generally attributed injury-induced knee in the intra-articular tissues during acute-phase ACL injury. degeneration to the long-term biomechanical changes in The objective of the present study was to elucidate the the microenvironment of the knee joint and have primarily effects of controlled abnormal joint movement on the focused on the long-term molecular kinetics in injured molecular biological response in intra-articular tissues ACLs [9, 10]. Consequently, many studies have shown that during the acute phase of ACL injury. We hypothesized that meniscal damage and chondral degeneration occur with controlling abnormal joint movement in a rat model would chronic ACL deficiency [11–13]. Furthermore, previous decrease the inflammatory response in intra-articular tis- studies have reported that ACL blood supply is poor [14] sues after the ACL injury acute phase. and he lack of a scaffold [15]. Therefore, ACL is recognized as a ligament difficult to heal after injury. Methods Surgical reconstruction treatment is the standard treat- Experimental design ment after ACL rupture. ACL reconstruction is the best All experiments were approved by the Saitama prefectural choice for athletes and/or high-level activity patients. University Animal Experiment Ethics Committee (permit However, conservative therapy after ACL injury is selected no. 24–2), and performed in accordance with their Guide- for patients with low and/or moderate activity levels, lines for the Care and Use of Laboratory Animals. children, elderly people. Although the ACL is not Eighty-four mature, 12-week-old Wistar male rats (body known to heal spontaneously in general [16], there are weight, 380–428 g, Japan SLC, Shizuoka, Japan) were many previous reports documenting spontaneous healing housed individually on a 12-h light-dark cycle with free of a ruptured ACL [15, 17–25]. Ihara et al. reported that access to food and water. The male rats were randomly 3-month conservative treatment resulted in a well-defined, assigned to the controlled abnormal movement (CAM) normal-sized, straight band in 74% of patients with groups, ACL-transection (ACL-T), sham-operated (SO), complete ACL rupture [23]. Moreover, many studies have or intact (IN), (each group, n = 21). The animals were not experimentally demonstrated the functional healing re- restricted activity until sacrifice. The room temperature sponses of injured ACLs [15, 17–19]. Extra-articular liga- was maintained at 23 °C ± 2 °C. To determine the inflam- ments such as the medial collateral ligament (MCL) exhibit matory response, the ACL and meniscus of the CAM a well-described healing response after injury in the absence group were histologically compared with those of the of surgical procedures [26]. Nguyen et al. showed that the other groups 1, 3, and 5 days after surgery (for 2 rats from human proximal 1/3 ACL has an intrinsic healing response each group); the inflammatory response was also com- with typical histological characteristics similar to those of pared across groups at 1, 3, and 5 days after surgery (for 5 the MCL [15]. Although these studies [15, 17–19]havere- rats from each group) (Fig. 1). ported that the ACL remnant has some possible functional healing responses that may induce spontaneous healing, Surgical procedure this theory has not been confirmed. Prior research showed that changing the joint kinematics A previous study demonstrated the effects of controlled of a knee with complete ruptured ACL results in the abnormal joint motion on modifying the intra-articular mo- down-regulation of inflammatory responses [33] and lecular response of ACL-ruptured knees, which led to leads to spontaneous healing [27]. These previous studies spontaneous ACL healing [27]. Although that study dem- used the CAM model used for the purpose of controlling onstrated a new mechanism of ACL healing, the molecular abnormal knee joint movement without restricted knee biological responses of the intra-articular tissues during the flexion (Fig. 2). Unlike casts, the CAM model restricts ACL healing process in the acute phase of injury remain only the anterior drawer of the tibia, therefore it does not Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 3 of 9 Day 0 ACL transection in the right knee SO group IN group ACL-T group CAM group No intervention ACL-T Surgical procedure Surgical procedure without ACL-T with ACL-T Day 1 Histological (n = 2 each) and biological (n = 5 each) evaluation: All groups Day 3 Day 5 Fig. 1 Flowchart showing the allocation of animals in the study. ACL-T, anterior cruciate ligament transection; CAM, controlled abnormal movement; IN, intact; SO, sham operated. Each group, n =21 inhibit the flexion of the knee joint. This model was de- of the tibial tuberosity in the mediolateral direction. Then, vised with reference to the brace used in previous study the skin was closed only in the ACL-T group. In the CAM [23]. These previous studies showed that controlling the group, a 2–0 nylon suture (Prolene, Ethicon Endo-Surgery motion of the knee and minimizing abnormal sagittal de- Japan) was passed through the tibial bone tunnel posterior viations between the femur and tibia within the range of to the condyle of the distal end of the femur and was then motion appropriate for ACL injury treatment leads to tied to the joint in order to prevent abnormal tibial trans- spontaneous healing [22, 23]. Therefore, in the present lation, according to the same procedure as that used in study, this CAM model was used to determine the effect previous study [27]. The nylon suture provided a directed of changing the intra-articular environment during the traction force to resist the anterior motion of the tibia with- acute phase of ACL injury. out restricted knee flexion (Fig. 2). After extra-articular The animals were anesthetized with pentobarbital braking, the skin was closed with running sutures. The SO (10 mg/kg) via intraperitoneal injections. The right knee group underwent skin and joint capsule incisions and underwent medial parapatellar arthrotomy, and the ACL tibial bone tunnel creation without ACL transection; was horizontally and completely transected at the mid- postoperatively, these rats were immediately allowed portion. After the ACL was transected, the joint capsule unrestricted movement. and parapatellar fascia were closed with a running suture using 4–0 Ethibond (Ethicon Endo-Surgery Japan, Tokyo, Japan), and a bone tunnel was created in the medial aspect Histological examination The intra-articular response in the acute phase of ACL injury was evaluated histologically at 1, 3, and 5 days, ac- cording to the same procedure as that used in previous study [27]. Two animals (each group) were sacrificed by exsanguination, and fixed in 4% paraformaldehyde after anesthetized with pentobarbital (10 mg/kg) via intraper- itoneal injections at 1, 3, and 5 days, and all tissues were decalcified in a 10% ethylenediaminetetraacetic acid-based solution (pH 7.4) at 4 °C for 5 to 6 weeks. After decalcified, the all tissues were infiltrated with phosphate buffered saline different containing sucrose at 4 °C (10%; 4 h, 15%; 4 h, and 20%;12 h), and embed- ded in an optimal cutting temperature compound (O.C.T., Sakura Finetek Japan, Tokyo, Japan). Longitudinal cryosections were cut along the sagittal plane with a thickness of 14 μm using cryostat (Leica 3050 S, Leica Microsystems AG, Wetzlar, Germany) and maintained Fig. 2 Extra-articular braking model. In the CAM group, a 2–0 nylon at − 80 °C. The cryosections were stained with suture was passed through the tibial bone tunnel posterior to the condyle of the distal end of the femur to control anterior tibial hematoxylin and eosin (H&E) in order to observe the translation and was then tied to the joint to prevent abnormal tibial microscopic morphological characteristics of the translation without restriction of knee flexion. ACL, anterior cruciate intra-articular response in the acute phase of ACL ligament. CAM, controlled abnormal movement injury. Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 4 of 9 Molecular biological evaluation gap of the ligament stump decreased (Fig. 3f and i). In the On postoperative days 1, 3, and 5, ACLs and menisci SO and IN groups, the ACL showed continuity based on were harvested from each group (n = 5) and evaluated the arrangement of the collagen fibers (Fig. 3g and h). gene expression related to the intra-articular reaction using real-time reverse transcription polymerase chain Expression of MMP-13 and TIMP-1 mRNA in intra-articular reaction (PCR). The tissue samples were homogenized, tissues after ACL injury and RNA was extracted using an Allprep DNA/RNA/ Significant differences in mRNA levels for MMP-13 in Protein mini kit (Qiagen, Hilden, Germany). Total RNA the ACL and medial meniscus were observed during the from each sample was reverse transcribed into comple- acute phase ACL injury between ACL-T and CAM groups mentary DNA (cDNA) using a high-capacity RNA to (p < 0.0001, Fig. 4a, b). Furthermore, the all intra-articular cDNA kit (Applied Biosystems, CA, USA), according to tissues showed a significantly higher expression on day 5 the same procedure as that used in previous study [27]. than day 1 in the ACL-T group (Fig. 4a–c). The CAM Real-time PCR was performed using a Chrome 4 Real-Time group showed a significantly higher expression on day 5 Detector (Bio-Rad Laboratories, Hercules, USA) with than day 1 only medial meniscus. On the other hands, TaqMan Gene Expression Assay probe inflammatory there was no significant difference in TIMP-1 mRNA ex- factors, matrix metalloproteinase-13 (MMP-13), and pression between the ACL-T group and the CAM group tissue inhibitor of metalloproteinase-1 (TIMP-1), ac- (p = 0.384, Fig. 4d–f). As with MMP-13 mRNA in the cording to the manufacturer’s instructions (Applied MMP-13/TIMP-1 ratio, a significant difference was ob- Biosystems). Beta-actin was selected as the reference served in the ACL and medial meniscus between ACL-T gene. The primers used are listed in Table 1 (TaqMan and CAM groups (p <0.01, Fig. 4g and h). In the lateral Gene Expression Assay, Applied Biosystems). meniscus, the MMP-13/TIMP-1 ratio was significantly Standard curves were established with standards pre- higher in the ACL-T group than that in the SO and IN pared from 1st standard cDNA (Genostaff, Tokyo, Japan) groups at each time point (p < 0.001 for all comparisons, for all primers. The transcript levels of the target genes Fig. 4i). The CAM group did not significantly differ from were normalized to beta-actin. the SO and IN groups with respect to the MMP-13/ TIMP-1 ratio at each time point. The SO and IN groups Statistical analysis showed significant differences in each gene expression in The experimental data are presented as the mean ± stand- any intra-articular tissues compared with the ACL-T and ard deviation (SD). Before analysis, the normal distribu- CAM groups (except the medial meniscus at day 1 and 3, tion of the data was confirmed using the Shapiro-Wilk the lateral meniscus at day 1in TIMP-1 mRNA, and lateral test. Gene expression was analyzed using two-way meniscus at each time point in MMP-13/TIMP-1 ratio). (group x time point) analysis of variance (ANOVA), with subsequent post hoc comparisons (via Bonferroni tests) used for comparisons among groups and time Discussion points (i.e., days 1, 3, and 5). A pvalue of less than The present study was to compare the intra-articular re- 0.05 was considered significant. Statistical analyses sponse between the CAM and ACL-T groups during the were performed using JMP statistical software, ver. 12.0 acute phase of ACL injury using histological and gene (SAS Institute, Inc., Cary, NC, USA). expression method. The primary results of the present study are the following: as compared with ACL-T group, Results CAM group exhibited (1) the gap of the ligament stump Influence of intra-articular tissues after ACL transection decreased, (2) significantly lower MMP-13/TIMP-1 ex- The ACL-T and CAM groups demonstrated cleavage of pression ratios. These findings partially supported our hy- the ACL at days 1, 3 and 5 (Fig. 3a–f). In the ACL-T pothesis; the inflammatory reaction in the intra-articular and CAM group, ACL cleavage could be confirmed in tissues decreased in the CAM group, and joint control all tissue (Fig. 3a–f and i). Five days after operation, hist- was associated with the inflammatory reaction during the ology showed widening of the gap of the ligament stump acute phase of ACL injury. in the ACL-T group (Fig. 3c). In the CAM group, the Our previous study demonstrated the effects of controlled abnormal joint motion on modifying the intra-articular molecular response of ACL ruptured knees, which led to Table 1 Gene expression assays used for real-time PCR spontaneous ACL healing [27]. Thus, our previous study Gene Assay number showed spontaneous ACL healing at 2 weeks postopera- Matrix metalloproteinase-13 (MMP-13) Rn01448194 tive using the controlled abnormal joint motion proced- Tissue inhibitor of metalloproteinase-1 (TIMP-1) Rn00580432 ure. In the present study, a rat model of this spontaneous Beta-actin Rn00667869_m1 healing was used to determine the effect of changing the Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 5 of 9 AB C DE F GH I Fig. 3 Histological evaluation in each group. These figures show longitudinal sections of the anterior cruciate ligament (ACL) stained with hematoxylin and eosin: (a) ACL-T group at day 1; (b) ACL-T group at day 3; (c) ACL-T group at day 5; (d) CAM group at day 1; (e) CAM group at day 3; (f, i) CAM group at day 5; (g) IN group; (h) SO group. The ACL-T and CAM groups demonstrated cleavage of the ACL at each time point (a–f). However, the gap in the ligament stump in the CAM group became very small, and fibroblast proliferation was observed around the stump (d–f, i). ACL-T, ACL-transection; CAM, controlled abnormal movement; IN, intact; SO, sham operated. Scale bars = 1 mm intra-articular environment in a CAM model during the issues with vascular supply [14], the lack of a scaffold acute phase of ACL injury. [15], and the lack of blood clot formation [35]). A previ- In the present study, controlled abnormal tibial trans- ous study showed that complete rupture of the ACL, lation led to a decrease in the regression of the ligament showing a “mop end” of the remnant in the inflammatory stump and a decrease in the inflammatory reaction in phase, led to gradual retraction of the ligament remnant the intra-articular tissues during acute-phase ACL in- [18]. These previous findings are in accordance with the jury. In contrast, the ACL-T group showed degeneration results of the present study showing that the ACL-T group of the ligament remnant. The difference between the exhibited retraction of the ligament remnant. However, ACL-T and CAM groups is the presence or absence of Ihara et al. showed that spontaneous healing of the ACL controlling abnormal joint movement. In general, failing occurs upon conservative treatment with early protective to control abnormal joint movement prevents healing mobilization after complete ACL rupture [22, 23]. Further- after ACL injury. A previous study reported that partial more, another previous studies reported that conservative ACL injuries cause 42% of patients to develop complete therapy (e.g., using specific brace) showed a significant im- ACL insufficiency [34]. Consequently, abnormal joint provement of anterior knee laxity comparable to patients movement was degenerative for the ACL remnant. Fur- treated with ACL reconstruction [21, 25]. These previous thermore, the poor healing capacity of the ACL has been studies pointed out that it is important to move articulation noted both experimentally and clinically (e.g., there are closer to normal and move it for protecting from early Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 6 of 9 AB C DE F GH I Fig. 4 Gene expression at days 1, 3, and 5 after surgery. Expression levels of matrix metalloproteinase-13 (MMP-13) in the anterior cruciate ligament (ACL, a), medial meniscus (b), and lateral meniscus (c). MMP-13 expression in the ACL and medial meniscus differed significantly between the ACL-T and CAM groups. The expression levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) in the ACL (d), medial meniscus (e), and lateral meniscus (f). TIMP-1 in all intra-articular tissues was not significantly different between the ACL-T and CAM groups. MMP-13/TIMP-1 ratios in the ACL (g), the medial meniscus (h), and the lateral meniscus (i). The ACL-T and CAM groups significantly differed with respect to the MMP-13/TIMP-1 ratio in the ACL and the medial meniscus on days 1 and 5 after surgery. On the other hand, in the lateral meniscus, the MMP-13/TIMP-1 ratio was significantly higher for the ACL-T group than for the other groups, with no significant difference between the CAM group and the SO and IN groups. ACL-T, ACL-transection; CAM, controlled abnormal movement; SO, sham operated, * p < 0.001 compared with day 1, † p < 0.001 compared with the ACL-T and CAM groups. Data showed mean ± standard deviation phase after injury. In addition, previous studies have Recent studies have focused on inflammation of the shown that controlling abnormal joint movement in a intra-articular tissues after ACL injury [17, 31]. The co- completely ruptured ACL results in the down-regulation ordinated expression of MMP-13 in intra-articular tis- of inflammatory responses [33] and leads to spontaneous sues and its accumulation in the synovial fluid may lead healing [27]. Furthermore, importance of weight-bearing to excessive matrix degeneration [31]. Moreover, in- has been pointed out in previous study [36]. The results of creased MMP-13 expression has been implicated in the present study also showed that an ACL remnant could osteoarthritis and rheumatoid arthritis [42]. Previous be maintained by controlling abnormal joint movement. studies have also reported that TIMP-1 specifically in- Therefore, controlling abnormal joint movement and hibits MMP-13 [43, 44], and the MMP-13/TIMP-1 ratio weight-bearing may contribute to a spontaneous healing is very important for tissue structure. In normal tissues, response of the ACL. Furthermore, in the case of ACL re- the stoichiometric MMP-13/TIMP-1 ratio is 1:1 [32]. An construction, the remnant is a very important factor. Pre- increase in MMP-13 is associated with the progression vious studies found that the preservation of the remnant of tissue destruction, and an increase of TIMP-1 is associ- tissue improved revascularization and remodeling of the ated with the progression of tissue fibrosis [45]. Compared graft and enhanced the biomechanical properties of the to the IN and SO groups, the ACL-T and CAM groups graft [37–41]. Consequently, controlling abnormal joint showed significantly higher expression levels of MMP-13 movement and weight-bearing after ACL injury is import- and TIMP-1 mRNA and a higher MMP-13/TIMP-1 ratio ant not only for conservative therapy but also for patients in all intra-articular tissues. Furthermore, the ACL-T group who undergo ACL reconstruction. showed significantly higher MMP-13 mRNA expression Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 7 of 9 compared with the CAM group. Thus, the results suggest to excision of the lateral meniscus [53]. Therefore, both that controlling abnormal joint movement after ACL injury the medial meniscus and the lateral meniscus have the inhibited MMP-13 mRNA expression in the intra-articular ability to brake the tibial front drawer. However, the med- tissues. Tang et al. reported that MMP-13 mRNA is ial and lateral menisci repeatedly receive mechanical stress expressed in the ACL and the intra-articular tissues during from abnormal joint movement after ACL injury. Degen- the acute phase of ACL injury [31]. Thepresent studysimi- eration of the medial and lateral menisci occurs after an larly showed that the MMP-13 mRNA expression levels ACL injury due to this repeated secondary mechanical were significantly higher in the intra-articular tissues after stress. In the present study, the MMP-13 mRNA expres- the acute phase of ACL injury. The ACL is located within sion of the medial meniscus and lateral meniscus was the articular capsule, and after injury, the ACL is exposed higher in the ACL-T group than in the CAM group due to to synovial fluid containing inflammatory substances that this secondary mechanical stress. inhibit healing [46]. Previous studies have also reported that The ACL is responsible for braking the forward move- the expression of MMP-13 mRNA significantly increases in ment of the tibia relative to the femur and for tibial rotation. the cartilage and synovium after ACL injury [31, 47, 48]. After ACL injury, this braking performance deteriorates, Therefore, it is thought that MMP-13 is expressed coopera- and the loss of this braking capacity in ACL injury patients tively by other intra-articular tissues, accumulates in the causes repeated damage to the articular cartilage and synovial fluid, and may promote ACL degeneration after in- meniscus. A previous study reported that more than jury. Accordingly, inhibiting the expression of MMP-13 in half of patients with an ACL injury suffer from second- the intra-articular tissues is considered an important factor ary knee OA [54]. The results of the present study sug- in inhibiting the degeneration of articular tissue. gested that the expression of MMP-13 mRNA in the The CAM group showed significantly lower MMP-13 acute phase of ACL injury was inhibited by controlling mRNA expression in the intra-articular tissues than did abnormal joint motion. This finding indicates a possible the ACL-T group. The differences in these groups was prevention strategy for joint degeneration after ACL in- the use suppression of abnormal movement of the tibia jury. A previous study reported that the occurrence of relative to the femur after ACL injury affected our re- additional knee injuries increased over time after ACL sults. Moreover, we showed that the mRNA expression injury, and the risk of additional meniscus injuries in- levels of TIMP-1, an inhibitor of MMP-13, increase as creased substantially 6 months after ACL injury [55]. the levels of MMP-13 mRNA increase, thereby inhibiting ACL reconstruction surgery is therefore recommended the activity of MMP-13 [49]. Because the expression of within 6 months after injury, but our findings suggest that TIMP-1 mRNA in the intra-articular tissues was not sig- degeneration of the joint has already occurred during the nificantly different between the ACL-T and CAM acute phase of ACL injury. Consequently, joint control groups, the ACL-T group showed a significantly higher during the acute phase is very important for patients with MMP-13/TIMP-1 ratio than the CAM group on day 5. reconstruction therapy after ACL injury. Thus, MMP-13 and TIMP-1 were not balanced in the The present study has several limitations. First, the in- ACL-T group. Moreover, a previous study also indicated vestigation was a small animal study, which limits the that degeneration, such as osseous tissue deterioration, generalizability of the results. The anatomical compo- results from the increased expression of MMP relative nents of humans and rats are similar; however, the joint to that of TIMP [50]. Therefore, more tissue degener- function of the knee and weight-bearing conditions are ation was assumed to have occurred in the ACL-T group different. These differences affect the joint kinematics of than in the CAM group based on the dynamics of the knee after ACL injury. Thus, the effect of controlled MMP-13 and TIMP-1. abnormal joint movement in the early phase of complete Previous studies have reported that meniscus injury ACL rupture may differ between humans and rats. Further- can occur due to mechanical stress from abnormal more, although we already performed the same surgical movement of the tibia after ACL injury [2, 50]. Allen et procedure in rabbit ACL, we cannot confirm for healing al. reported increases in the front drawer of the tibia and ACL in the rabbit. We considered that the difference in the amount of the load response to the medial meniscus walking style between rabbits and rats is affecting. Although after ACL injury [51]. Levy et al. reported that the med- rats walk for four legs, walk alternately like humans. On the ial meniscus contributes to the braking of the tibial front other hands, rabbit moves forward by kicking the ground drawer after ACL injury [52]. Therefore, mechanical with both legs. Therefore, it is necessary to select animals stress to the medial meniscus increases after ACL injury. with four leg walking (e.g., pig and/or dog) in future The lateral meniscus is an important component in the research. Second, we studied the healing process of a braking of the tibial front drawer. Musahl et al. reported completely injured ACL only during the early phase that the amount of forward displacement significantly (i.e., until post-injury day 5). Third, this study was per- increased compared to the amount of displacement prior formed a small sample. Therefore, statistical differences Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 8 of 9 could not be considered in histologic examination. In the Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in future, it is necessary to quantitatively present the narrow- published maps and institutional affiliations. ing of the ACL stump by increasing the number of sam- ples. Fourth, in the molecular biology evaluation of the Author details Department of Neuroscience and Therapeutics, Hiroshima University present study, we examined only MMP-13 and TIMP-1. Graduate School of Biomedical and Health Sciences, Hiroshima, Japan. Research has shown that inflammatory factors such as 2 Division of Rehabilitation, Department of Clinical Practice and Support, cytokines (e.g., interleukin (IL)-6 and IL-8) are present Hiroshima University Hospital, Hiroshima, Japan. Department of Physical Therapy, School of Health and Social Services, Saitama Prefectural University, at elevated levels in synovial fluid during the acute 820 Sannomiya, Koshigaya-shi, Saitama 343-8540, Japan. Sakai City Medical phase of ACL injury [28]. Therefore, it will be ideal to Center, Osaka, Japan. evaluate inflammatory factors in addition to MMPs and Received: 25 January 2018 Accepted: 21 May 2018 TIMPs. Finally, our method for rupturing the ACL was unlike the common mechanism of ACL injury in humans. Further long-term studies using different animals, other References inflammatory factors, and another method of ACL rup- 1. Butler DL, Noyes FR, Grood ES. 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Effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of anterior cruciate ligament injury in a rat model

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Medicine & Public Health; Orthopedics; Rehabilitation; Rheumatology; Sports Medicine; Internal Medicine; Epidemiology
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Abstract

Background: The anterior cruciate ligament (ACL) is responsible for braking forward movement of the tibia relative to the femur and for tibial rotation. After ACL injury, this braking performance deteriorates, inducing abnormal joint movement. The purpose of this study was to clarify the effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of ACL injury. Methods: Eighty-four mature Wistar male rats were randomly assigned to a controlled abnormal movement (CAM) group, an ACL-transection (ACL-T) group, a sham-operated group, or an intact group. The ACL was completely transected at its midportion in the ACL-T and CAM groups, and a nylon suture was used to control abnormal tibial translation in the CAM group. The sham-operated group underwent skin and joint capsule incisions and tibial drilling without ACL transection. Animals were not restricted activity until sacrifice 1, 3, or 5 days after surgery for histological and gene expression assessments. Acute-phase inflammation requires an important balance between degenerative and biosynthetic processes and is controlled by the activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Both types of gene were analyzed in this study. Results: The ACL-T and CAM groups exhibited cleavage of the ACL at all time points. However, for the CAM group, the gap in the ligament stump was extremely small, and fibroblast proliferation was observed around the stump. Relative to the ACL-T group, the CAM group demonstrated significantly lower expression of MMP-13 mRNA and a lower MMP-13/TIMP-1 ratio on days 1 and 5 in the ACL, the medial meniscus and the lateral meniscus. The expression of TIMP-1 mRNA was not significantly different between the ACL-T and CAM groups. Conclusions: The study results suggested that controlling abnormal movement inhibited the inflammatory reaction in intra-articular tissues after ACL injury. This reaction was down-regulated in intra-articular tissues in the CAM group. Abnormal joint control caused prolonged inflammation and inhibited remodeling during the acute phase of ACL rupture. Keywords: Anterior cruciate ligament, Matrix metalloproteinase-13, Conservative therapy, Acute phase injury * Correspondence: takayanagi-kiyomi@spu.ac.jp Department of Physical Therapy, School of Health and Social Services, Saitama Prefectural University, 820 Sannomiya, Koshigaya-shi, Saitama 343-8540, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 2 of 9 Background unclear. It is thought that controlled abnormal joint move- The anterior cruciate ligament (ACL) plays an important ment is an important factor for spontaneous ACL healing. role in controlling and stabilizing the knee joint; it is the Many previous studies have reported the molecular primary restraint against anterior tibial translation [1]. biological responses in the intra-articular tissues dur- The prevalence of injury to the ACL is quite high, particu- ing the acute phase [28–31]. However, to the best of larly among athletes who perform pivoting activities [2, 3]. the authors’ knowledge, no reports have yet focused on the Furthermore, previous reported that female subjects signifi- effects of abnormal joint movement on intra-articular tis- cantly higher risk of ACL injury than male subjects [4, 5]. sues. The acute-phase inflammatory response plays an im- Treatment after ACL injury often involves reconstructive portant role in the wound healing process, and the balance surgery, which is performed on more than half of ACL in- between the degenerative and biosynthetic arms of this jury patients [6, 7]. process is controlled by the activities of matrix metallopro- ACL deficiency can induce the degeneration of other teinases (MMPs) and tissue inhibitors of metalloproteinases intra-articular tissues (i.e., cartilage and meniscus), which is (TIMPs) [32]. In the present study, we focused on MMPs a risk factor for the development of osteoarthritis [8]. Previ- and TIMPs to determine the molecular biological response ous studies have generally attributed injury-induced knee in the intra-articular tissues during acute-phase ACL injury. degeneration to the long-term biomechanical changes in The objective of the present study was to elucidate the the microenvironment of the knee joint and have primarily effects of controlled abnormal joint movement on the focused on the long-term molecular kinetics in injured molecular biological response in intra-articular tissues ACLs [9, 10]. Consequently, many studies have shown that during the acute phase of ACL injury. We hypothesized that meniscal damage and chondral degeneration occur with controlling abnormal joint movement in a rat model would chronic ACL deficiency [11–13]. Furthermore, previous decrease the inflammatory response in intra-articular tis- studies have reported that ACL blood supply is poor [14] sues after the ACL injury acute phase. and he lack of a scaffold [15]. Therefore, ACL is recognized as a ligament difficult to heal after injury. Methods Surgical reconstruction treatment is the standard treat- Experimental design ment after ACL rupture. ACL reconstruction is the best All experiments were approved by the Saitama prefectural choice for athletes and/or high-level activity patients. University Animal Experiment Ethics Committee (permit However, conservative therapy after ACL injury is selected no. 24–2), and performed in accordance with their Guide- for patients with low and/or moderate activity levels, lines for the Care and Use of Laboratory Animals. children, elderly people. Although the ACL is not Eighty-four mature, 12-week-old Wistar male rats (body known to heal spontaneously in general [16], there are weight, 380–428 g, Japan SLC, Shizuoka, Japan) were many previous reports documenting spontaneous healing housed individually on a 12-h light-dark cycle with free of a ruptured ACL [15, 17–25]. Ihara et al. reported that access to food and water. The male rats were randomly 3-month conservative treatment resulted in a well-defined, assigned to the controlled abnormal movement (CAM) normal-sized, straight band in 74% of patients with groups, ACL-transection (ACL-T), sham-operated (SO), complete ACL rupture [23]. Moreover, many studies have or intact (IN), (each group, n = 21). The animals were not experimentally demonstrated the functional healing re- restricted activity until sacrifice. The room temperature sponses of injured ACLs [15, 17–19]. Extra-articular liga- was maintained at 23 °C ± 2 °C. To determine the inflam- ments such as the medial collateral ligament (MCL) exhibit matory response, the ACL and meniscus of the CAM a well-described healing response after injury in the absence group were histologically compared with those of the of surgical procedures [26]. Nguyen et al. showed that the other groups 1, 3, and 5 days after surgery (for 2 rats from human proximal 1/3 ACL has an intrinsic healing response each group); the inflammatory response was also com- with typical histological characteristics similar to those of pared across groups at 1, 3, and 5 days after surgery (for 5 the MCL [15]. Although these studies [15, 17–19]havere- rats from each group) (Fig. 1). ported that the ACL remnant has some possible functional healing responses that may induce spontaneous healing, Surgical procedure this theory has not been confirmed. Prior research showed that changing the joint kinematics A previous study demonstrated the effects of controlled of a knee with complete ruptured ACL results in the abnormal joint motion on modifying the intra-articular mo- down-regulation of inflammatory responses [33] and lecular response of ACL-ruptured knees, which led to leads to spontaneous healing [27]. These previous studies spontaneous ACL healing [27]. Although that study dem- used the CAM model used for the purpose of controlling onstrated a new mechanism of ACL healing, the molecular abnormal knee joint movement without restricted knee biological responses of the intra-articular tissues during the flexion (Fig. 2). Unlike casts, the CAM model restricts ACL healing process in the acute phase of injury remain only the anterior drawer of the tibia, therefore it does not Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 3 of 9 Day 0 ACL transection in the right knee SO group IN group ACL-T group CAM group No intervention ACL-T Surgical procedure Surgical procedure without ACL-T with ACL-T Day 1 Histological (n = 2 each) and biological (n = 5 each) evaluation: All groups Day 3 Day 5 Fig. 1 Flowchart showing the allocation of animals in the study. ACL-T, anterior cruciate ligament transection; CAM, controlled abnormal movement; IN, intact; SO, sham operated. Each group, n =21 inhibit the flexion of the knee joint. This model was de- of the tibial tuberosity in the mediolateral direction. Then, vised with reference to the brace used in previous study the skin was closed only in the ACL-T group. In the CAM [23]. These previous studies showed that controlling the group, a 2–0 nylon suture (Prolene, Ethicon Endo-Surgery motion of the knee and minimizing abnormal sagittal de- Japan) was passed through the tibial bone tunnel posterior viations between the femur and tibia within the range of to the condyle of the distal end of the femur and was then motion appropriate for ACL injury treatment leads to tied to the joint in order to prevent abnormal tibial trans- spontaneous healing [22, 23]. Therefore, in the present lation, according to the same procedure as that used in study, this CAM model was used to determine the effect previous study [27]. The nylon suture provided a directed of changing the intra-articular environment during the traction force to resist the anterior motion of the tibia with- acute phase of ACL injury. out restricted knee flexion (Fig. 2). After extra-articular The animals were anesthetized with pentobarbital braking, the skin was closed with running sutures. The SO (10 mg/kg) via intraperitoneal injections. The right knee group underwent skin and joint capsule incisions and underwent medial parapatellar arthrotomy, and the ACL tibial bone tunnel creation without ACL transection; was horizontally and completely transected at the mid- postoperatively, these rats were immediately allowed portion. After the ACL was transected, the joint capsule unrestricted movement. and parapatellar fascia were closed with a running suture using 4–0 Ethibond (Ethicon Endo-Surgery Japan, Tokyo, Japan), and a bone tunnel was created in the medial aspect Histological examination The intra-articular response in the acute phase of ACL injury was evaluated histologically at 1, 3, and 5 days, ac- cording to the same procedure as that used in previous study [27]. Two animals (each group) were sacrificed by exsanguination, and fixed in 4% paraformaldehyde after anesthetized with pentobarbital (10 mg/kg) via intraper- itoneal injections at 1, 3, and 5 days, and all tissues were decalcified in a 10% ethylenediaminetetraacetic acid-based solution (pH 7.4) at 4 °C for 5 to 6 weeks. After decalcified, the all tissues were infiltrated with phosphate buffered saline different containing sucrose at 4 °C (10%; 4 h, 15%; 4 h, and 20%;12 h), and embed- ded in an optimal cutting temperature compound (O.C.T., Sakura Finetek Japan, Tokyo, Japan). Longitudinal cryosections were cut along the sagittal plane with a thickness of 14 μm using cryostat (Leica 3050 S, Leica Microsystems AG, Wetzlar, Germany) and maintained Fig. 2 Extra-articular braking model. In the CAM group, a 2–0 nylon at − 80 °C. The cryosections were stained with suture was passed through the tibial bone tunnel posterior to the condyle of the distal end of the femur to control anterior tibial hematoxylin and eosin (H&E) in order to observe the translation and was then tied to the joint to prevent abnormal tibial microscopic morphological characteristics of the translation without restriction of knee flexion. ACL, anterior cruciate intra-articular response in the acute phase of ACL ligament. CAM, controlled abnormal movement injury. Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 4 of 9 Molecular biological evaluation gap of the ligament stump decreased (Fig. 3f and i). In the On postoperative days 1, 3, and 5, ACLs and menisci SO and IN groups, the ACL showed continuity based on were harvested from each group (n = 5) and evaluated the arrangement of the collagen fibers (Fig. 3g and h). gene expression related to the intra-articular reaction using real-time reverse transcription polymerase chain Expression of MMP-13 and TIMP-1 mRNA in intra-articular reaction (PCR). The tissue samples were homogenized, tissues after ACL injury and RNA was extracted using an Allprep DNA/RNA/ Significant differences in mRNA levels for MMP-13 in Protein mini kit (Qiagen, Hilden, Germany). Total RNA the ACL and medial meniscus were observed during the from each sample was reverse transcribed into comple- acute phase ACL injury between ACL-T and CAM groups mentary DNA (cDNA) using a high-capacity RNA to (p < 0.0001, Fig. 4a, b). Furthermore, the all intra-articular cDNA kit (Applied Biosystems, CA, USA), according to tissues showed a significantly higher expression on day 5 the same procedure as that used in previous study [27]. than day 1 in the ACL-T group (Fig. 4a–c). The CAM Real-time PCR was performed using a Chrome 4 Real-Time group showed a significantly higher expression on day 5 Detector (Bio-Rad Laboratories, Hercules, USA) with than day 1 only medial meniscus. On the other hands, TaqMan Gene Expression Assay probe inflammatory there was no significant difference in TIMP-1 mRNA ex- factors, matrix metalloproteinase-13 (MMP-13), and pression between the ACL-T group and the CAM group tissue inhibitor of metalloproteinase-1 (TIMP-1), ac- (p = 0.384, Fig. 4d–f). As with MMP-13 mRNA in the cording to the manufacturer’s instructions (Applied MMP-13/TIMP-1 ratio, a significant difference was ob- Biosystems). Beta-actin was selected as the reference served in the ACL and medial meniscus between ACL-T gene. The primers used are listed in Table 1 (TaqMan and CAM groups (p <0.01, Fig. 4g and h). In the lateral Gene Expression Assay, Applied Biosystems). meniscus, the MMP-13/TIMP-1 ratio was significantly Standard curves were established with standards pre- higher in the ACL-T group than that in the SO and IN pared from 1st standard cDNA (Genostaff, Tokyo, Japan) groups at each time point (p < 0.001 for all comparisons, for all primers. The transcript levels of the target genes Fig. 4i). The CAM group did not significantly differ from were normalized to beta-actin. the SO and IN groups with respect to the MMP-13/ TIMP-1 ratio at each time point. The SO and IN groups Statistical analysis showed significant differences in each gene expression in The experimental data are presented as the mean ± stand- any intra-articular tissues compared with the ACL-T and ard deviation (SD). Before analysis, the normal distribu- CAM groups (except the medial meniscus at day 1 and 3, tion of the data was confirmed using the Shapiro-Wilk the lateral meniscus at day 1in TIMP-1 mRNA, and lateral test. Gene expression was analyzed using two-way meniscus at each time point in MMP-13/TIMP-1 ratio). (group x time point) analysis of variance (ANOVA), with subsequent post hoc comparisons (via Bonferroni tests) used for comparisons among groups and time Discussion points (i.e., days 1, 3, and 5). A pvalue of less than The present study was to compare the intra-articular re- 0.05 was considered significant. Statistical analyses sponse between the CAM and ACL-T groups during the were performed using JMP statistical software, ver. 12.0 acute phase of ACL injury using histological and gene (SAS Institute, Inc., Cary, NC, USA). expression method. The primary results of the present study are the following: as compared with ACL-T group, Results CAM group exhibited (1) the gap of the ligament stump Influence of intra-articular tissues after ACL transection decreased, (2) significantly lower MMP-13/TIMP-1 ex- The ACL-T and CAM groups demonstrated cleavage of pression ratios. These findings partially supported our hy- the ACL at days 1, 3 and 5 (Fig. 3a–f). In the ACL-T pothesis; the inflammatory reaction in the intra-articular and CAM group, ACL cleavage could be confirmed in tissues decreased in the CAM group, and joint control all tissue (Fig. 3a–f and i). Five days after operation, hist- was associated with the inflammatory reaction during the ology showed widening of the gap of the ligament stump acute phase of ACL injury. in the ACL-T group (Fig. 3c). In the CAM group, the Our previous study demonstrated the effects of controlled abnormal joint motion on modifying the intra-articular molecular response of ACL ruptured knees, which led to Table 1 Gene expression assays used for real-time PCR spontaneous ACL healing [27]. Thus, our previous study Gene Assay number showed spontaneous ACL healing at 2 weeks postopera- Matrix metalloproteinase-13 (MMP-13) Rn01448194 tive using the controlled abnormal joint motion proced- Tissue inhibitor of metalloproteinase-1 (TIMP-1) Rn00580432 ure. In the present study, a rat model of this spontaneous Beta-actin Rn00667869_m1 healing was used to determine the effect of changing the Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 5 of 9 AB C DE F GH I Fig. 3 Histological evaluation in each group. These figures show longitudinal sections of the anterior cruciate ligament (ACL) stained with hematoxylin and eosin: (a) ACL-T group at day 1; (b) ACL-T group at day 3; (c) ACL-T group at day 5; (d) CAM group at day 1; (e) CAM group at day 3; (f, i) CAM group at day 5; (g) IN group; (h) SO group. The ACL-T and CAM groups demonstrated cleavage of the ACL at each time point (a–f). However, the gap in the ligament stump in the CAM group became very small, and fibroblast proliferation was observed around the stump (d–f, i). ACL-T, ACL-transection; CAM, controlled abnormal movement; IN, intact; SO, sham operated. Scale bars = 1 mm intra-articular environment in a CAM model during the issues with vascular supply [14], the lack of a scaffold acute phase of ACL injury. [15], and the lack of blood clot formation [35]). A previ- In the present study, controlled abnormal tibial trans- ous study showed that complete rupture of the ACL, lation led to a decrease in the regression of the ligament showing a “mop end” of the remnant in the inflammatory stump and a decrease in the inflammatory reaction in phase, led to gradual retraction of the ligament remnant the intra-articular tissues during acute-phase ACL in- [18]. These previous findings are in accordance with the jury. In contrast, the ACL-T group showed degeneration results of the present study showing that the ACL-T group of the ligament remnant. The difference between the exhibited retraction of the ligament remnant. However, ACL-T and CAM groups is the presence or absence of Ihara et al. showed that spontaneous healing of the ACL controlling abnormal joint movement. In general, failing occurs upon conservative treatment with early protective to control abnormal joint movement prevents healing mobilization after complete ACL rupture [22, 23]. Further- after ACL injury. A previous study reported that partial more, another previous studies reported that conservative ACL injuries cause 42% of patients to develop complete therapy (e.g., using specific brace) showed a significant im- ACL insufficiency [34]. Consequently, abnormal joint provement of anterior knee laxity comparable to patients movement was degenerative for the ACL remnant. Fur- treated with ACL reconstruction [21, 25]. These previous thermore, the poor healing capacity of the ACL has been studies pointed out that it is important to move articulation noted both experimentally and clinically (e.g., there are closer to normal and move it for protecting from early Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 6 of 9 AB C DE F GH I Fig. 4 Gene expression at days 1, 3, and 5 after surgery. Expression levels of matrix metalloproteinase-13 (MMP-13) in the anterior cruciate ligament (ACL, a), medial meniscus (b), and lateral meniscus (c). MMP-13 expression in the ACL and medial meniscus differed significantly between the ACL-T and CAM groups. The expression levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) in the ACL (d), medial meniscus (e), and lateral meniscus (f). TIMP-1 in all intra-articular tissues was not significantly different between the ACL-T and CAM groups. MMP-13/TIMP-1 ratios in the ACL (g), the medial meniscus (h), and the lateral meniscus (i). The ACL-T and CAM groups significantly differed with respect to the MMP-13/TIMP-1 ratio in the ACL and the medial meniscus on days 1 and 5 after surgery. On the other hand, in the lateral meniscus, the MMP-13/TIMP-1 ratio was significantly higher for the ACL-T group than for the other groups, with no significant difference between the CAM group and the SO and IN groups. ACL-T, ACL-transection; CAM, controlled abnormal movement; SO, sham operated, * p < 0.001 compared with day 1, † p < 0.001 compared with the ACL-T and CAM groups. Data showed mean ± standard deviation phase after injury. In addition, previous studies have Recent studies have focused on inflammation of the shown that controlling abnormal joint movement in a intra-articular tissues after ACL injury [17, 31]. The co- completely ruptured ACL results in the down-regulation ordinated expression of MMP-13 in intra-articular tis- of inflammatory responses [33] and leads to spontaneous sues and its accumulation in the synovial fluid may lead healing [27]. Furthermore, importance of weight-bearing to excessive matrix degeneration [31]. Moreover, in- has been pointed out in previous study [36]. The results of creased MMP-13 expression has been implicated in the present study also showed that an ACL remnant could osteoarthritis and rheumatoid arthritis [42]. Previous be maintained by controlling abnormal joint movement. studies have also reported that TIMP-1 specifically in- Therefore, controlling abnormal joint movement and hibits MMP-13 [43, 44], and the MMP-13/TIMP-1 ratio weight-bearing may contribute to a spontaneous healing is very important for tissue structure. In normal tissues, response of the ACL. Furthermore, in the case of ACL re- the stoichiometric MMP-13/TIMP-1 ratio is 1:1 [32]. An construction, the remnant is a very important factor. Pre- increase in MMP-13 is associated with the progression vious studies found that the preservation of the remnant of tissue destruction, and an increase of TIMP-1 is associ- tissue improved revascularization and remodeling of the ated with the progression of tissue fibrosis [45]. Compared graft and enhanced the biomechanical properties of the to the IN and SO groups, the ACL-T and CAM groups graft [37–41]. Consequently, controlling abnormal joint showed significantly higher expression levels of MMP-13 movement and weight-bearing after ACL injury is import- and TIMP-1 mRNA and a higher MMP-13/TIMP-1 ratio ant not only for conservative therapy but also for patients in all intra-articular tissues. Furthermore, the ACL-T group who undergo ACL reconstruction. showed significantly higher MMP-13 mRNA expression Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 7 of 9 compared with the CAM group. Thus, the results suggest to excision of the lateral meniscus [53]. Therefore, both that controlling abnormal joint movement after ACL injury the medial meniscus and the lateral meniscus have the inhibited MMP-13 mRNA expression in the intra-articular ability to brake the tibial front drawer. However, the med- tissues. Tang et al. reported that MMP-13 mRNA is ial and lateral menisci repeatedly receive mechanical stress expressed in the ACL and the intra-articular tissues during from abnormal joint movement after ACL injury. Degen- the acute phase of ACL injury [31]. Thepresent studysimi- eration of the medial and lateral menisci occurs after an larly showed that the MMP-13 mRNA expression levels ACL injury due to this repeated secondary mechanical were significantly higher in the intra-articular tissues after stress. In the present study, the MMP-13 mRNA expres- the acute phase of ACL injury. The ACL is located within sion of the medial meniscus and lateral meniscus was the articular capsule, and after injury, the ACL is exposed higher in the ACL-T group than in the CAM group due to to synovial fluid containing inflammatory substances that this secondary mechanical stress. inhibit healing [46]. Previous studies have also reported that The ACL is responsible for braking the forward move- the expression of MMP-13 mRNA significantly increases in ment of the tibia relative to the femur and for tibial rotation. the cartilage and synovium after ACL injury [31, 47, 48]. After ACL injury, this braking performance deteriorates, Therefore, it is thought that MMP-13 is expressed coopera- and the loss of this braking capacity in ACL injury patients tively by other intra-articular tissues, accumulates in the causes repeated damage to the articular cartilage and synovial fluid, and may promote ACL degeneration after in- meniscus. A previous study reported that more than jury. Accordingly, inhibiting the expression of MMP-13 in half of patients with an ACL injury suffer from second- the intra-articular tissues is considered an important factor ary knee OA [54]. The results of the present study sug- in inhibiting the degeneration of articular tissue. gested that the expression of MMP-13 mRNA in the The CAM group showed significantly lower MMP-13 acute phase of ACL injury was inhibited by controlling mRNA expression in the intra-articular tissues than did abnormal joint motion. This finding indicates a possible the ACL-T group. The differences in these groups was prevention strategy for joint degeneration after ACL in- the use suppression of abnormal movement of the tibia jury. A previous study reported that the occurrence of relative to the femur after ACL injury affected our re- additional knee injuries increased over time after ACL sults. Moreover, we showed that the mRNA expression injury, and the risk of additional meniscus injuries in- levels of TIMP-1, an inhibitor of MMP-13, increase as creased substantially 6 months after ACL injury [55]. the levels of MMP-13 mRNA increase, thereby inhibiting ACL reconstruction surgery is therefore recommended the activity of MMP-13 [49]. Because the expression of within 6 months after injury, but our findings suggest that TIMP-1 mRNA in the intra-articular tissues was not sig- degeneration of the joint has already occurred during the nificantly different between the ACL-T and CAM acute phase of ACL injury. Consequently, joint control groups, the ACL-T group showed a significantly higher during the acute phase is very important for patients with MMP-13/TIMP-1 ratio than the CAM group on day 5. reconstruction therapy after ACL injury. Thus, MMP-13 and TIMP-1 were not balanced in the The present study has several limitations. First, the in- ACL-T group. Moreover, a previous study also indicated vestigation was a small animal study, which limits the that degeneration, such as osseous tissue deterioration, generalizability of the results. The anatomical compo- results from the increased expression of MMP relative nents of humans and rats are similar; however, the joint to that of TIMP [50]. Therefore, more tissue degener- function of the knee and weight-bearing conditions are ation was assumed to have occurred in the ACL-T group different. These differences affect the joint kinematics of than in the CAM group based on the dynamics of the knee after ACL injury. Thus, the effect of controlled MMP-13 and TIMP-1. abnormal joint movement in the early phase of complete Previous studies have reported that meniscus injury ACL rupture may differ between humans and rats. Further- can occur due to mechanical stress from abnormal more, although we already performed the same surgical movement of the tibia after ACL injury [2, 50]. Allen et procedure in rabbit ACL, we cannot confirm for healing al. reported increases in the front drawer of the tibia and ACL in the rabbit. We considered that the difference in the amount of the load response to the medial meniscus walking style between rabbits and rats is affecting. Although after ACL injury [51]. Levy et al. reported that the med- rats walk for four legs, walk alternately like humans. On the ial meniscus contributes to the braking of the tibial front other hands, rabbit moves forward by kicking the ground drawer after ACL injury [52]. Therefore, mechanical with both legs. Therefore, it is necessary to select animals stress to the medial meniscus increases after ACL injury. with four leg walking (e.g., pig and/or dog) in future The lateral meniscus is an important component in the research. Second, we studied the healing process of a braking of the tibial front drawer. Musahl et al. reported completely injured ACL only during the early phase that the amount of forward displacement significantly (i.e., until post-injury day 5). Third, this study was per- increased compared to the amount of displacement prior formed a small sample. Therefore, statistical differences Nishikawa et al. BMC Musculoskeletal Disorders (2018) 19:175 Page 8 of 9 could not be considered in histologic examination. In the Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in future, it is necessary to quantitatively present the narrow- published maps and institutional affiliations. ing of the ACL stump by increasing the number of sam- ples. Fourth, in the molecular biology evaluation of the Author details Department of Neuroscience and Therapeutics, Hiroshima University present study, we examined only MMP-13 and TIMP-1. Graduate School of Biomedical and Health Sciences, Hiroshima, Japan. Research has shown that inflammatory factors such as 2 Division of Rehabilitation, Department of Clinical Practice and Support, cytokines (e.g., interleukin (IL)-6 and IL-8) are present Hiroshima University Hospital, Hiroshima, Japan. Department of Physical Therapy, School of Health and Social Services, Saitama Prefectural University, at elevated levels in synovial fluid during the acute 820 Sannomiya, Koshigaya-shi, Saitama 343-8540, Japan. Sakai City Medical phase of ACL injury [28]. Therefore, it will be ideal to Center, Osaka, Japan. evaluate inflammatory factors in addition to MMPs and Received: 25 January 2018 Accepted: 21 May 2018 TIMPs. Finally, our method for rupturing the ACL was unlike the common mechanism of ACL injury in humans. Further long-term studies using different animals, other References inflammatory factors, and another method of ACL rup- 1. Butler DL, Noyes FR, Grood ES. 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BMC Musculoskeletal DisordersSpringer Journals

Published: May 29, 2018

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