Background
The anterior cruciate ligament (ACL) plays an important role in controlling and stabilizing the knee joint; it is the primary restraint against anterior tibial translation [
1]. The prevalence of injury to the ACL is quite high, particularly among athletes who perform pivoting activities [
2,
3]. Furthermore, previous reported that female subjects significantly higher risk of ACL injury than male subjects [
4,
5]. Treatment after ACL injury often involves reconstructive surgery, which is performed on more than half of ACL injury patients [
6,
7].
ACL deficiency can induce the degeneration of other intra-articular tissues (i.e., cartilage and meniscus), which is a risk factor for the development of osteoarthritis [
8]. Previous studies have generally attributed injury-induced knee degeneration to the long-term biomechanical changes in the microenvironment of the knee joint and have primarily focused on the long-term molecular kinetics in injured ACLs [
9,
10]. Consequently, many studies have shown that meniscal damage and chondral degeneration occur with chronic ACL deficiency [
11‐
13]. Furthermore, previous studies have reported that ACL blood supply is poor [
14] and he lack of a scaffold [
15]. Therefore, ACL is recognized as a ligament difficult to heal after injury.
Surgical reconstruction treatment is the standard treatment after ACL rupture. ACL reconstruction is the best choice for athletes and/or high-level activity patients. However, conservative therapy after ACL injury is selected for patients with low and/or moderate activity levels, children, elderly people. Although the ACL is not known to heal spontaneously in general [
16], there are many previous reports documenting spontaneous healing of a ruptured ACL [
15,
17‐
25]. Ihara et al. reported that 3-month conservative treatment resulted in a well-defined, normal-sized, straight band in 74% of patients with complete ACL rupture [
23]. Moreover, many studies have experimentally demonstrated the functional healing responses of injured ACLs [
15,
17‐
19]. Extra-articular ligaments such as the medial collateral ligament (MCL) exhibit a well-described healing response after injury in the absence of surgical procedures [
26]. Nguyen et al. showed that the human proximal 1/3 ACL has an intrinsic healing response with typical histological characteristics similar to those of the MCL [
15]. Although these studies [
15,
17‐
19] have reported that the ACL remnant has some possible functional healing responses that may induce spontaneous healing, this theory has not been confirmed.
A previous study demonstrated the effects of controlled abnormal joint motion on modifying the intra-articular molecular response of ACL-ruptured knees, which led to spontaneous ACL healing [
27]. Although that study demonstrated a new mechanism of ACL healing, the molecular biological responses of the intra-articular tissues during the ACL healing process in the acute phase of injury remain unclear. It is thought that controlled abnormal joint movement is an important factor for spontaneous ACL healing. Many previous studies have reported the molecular biological responses in the intra-articular tissues during the acute phase [
28‐
31]. However, to the best of the authors’ knowledge, no reports have yet focused on the effects of abnormal joint movement on intra-articular tissues. The acute-phase inflammatory response plays an important role in the wound healing process, and the balance between the degenerative and biosynthetic arms of this process is controlled by the activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [
32]. In the present study, we focused on MMPs and TIMPs to determine the molecular biological response in the intra-articular tissues during acute-phase ACL injury.
The objective of the present study was to elucidate the effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of ACL injury. We hypothesized that controlling abnormal joint movement in a rat model would decrease the inflammatory response in intra-articular tissues after the ACL injury acute phase.
Discussion
The present study was to compare the intra-articular response between the CAM and ACL-T groups during the acute phase of ACL injury using histological and gene expression method. The primary results of the present study are the following: as compared with ACL-T group, CAM group exhibited (1) the gap of the ligament stump decreased, (2) significantly lower MMP-13/TIMP-1 expression ratios. These findings partially supported our hypothesis; the inflammatory reaction in the intra-articular tissues decreased in the CAM group, and joint control was associated with the inflammatory reaction during the acute phase of ACL injury.
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 spontaneous ACL healing [
27]. Thus, our previous study showed spontaneous ACL healing at 2 weeks postoperative using the controlled abnormal joint motion procedure. In the present study, a rat model of this spontaneous healing was used to determine the effect of changing the intra-articular environment in a CAM model during the acute phase of ACL injury.
In the present study, controlled abnormal tibial translation led to a decrease in the regression of the ligament stump and a decrease in the inflammatory reaction in the intra-articular tissues during acute-phase ACL injury. In contrast, the ACL-T group showed degeneration of the ligament remnant. The difference between the ACL-T and CAM groups is the presence or absence of controlling abnormal joint movement. In general, failing to control abnormal joint movement prevents healing after ACL injury. A previous study reported that partial ACL injuries cause 42% of patients to develop complete ACL insufficiency [
34]. Consequently, abnormal joint movement was degenerative for the ACL remnant. Furthermore, the poor healing capacity of the ACL has been noted both experimentally and clinically (e.g., there are issues with vascular supply [
14], the lack of a scaffold [
15], and the lack of blood clot formation [
35]). A previous study showed that complete rupture of the ACL, showing a “mop end” of the remnant in the inflammatory phase, led to gradual retraction of the ligament remnant [
18]. These previous findings are in accordance with the results of the present study showing that the ACL-T group exhibited retraction of the ligament remnant. However, Ihara et al. showed that spontaneous healing of the ACL occurs upon conservative treatment with early protective mobilization after complete ACL rupture [
22,
23]. Furthermore, another previous studies reported that conservative therapy (e.g., using specific brace) showed a significant improvement of anterior knee laxity comparable to patients treated with ACL reconstruction [
21,
25]. These previous studies pointed out that it is important to move articulation closer to normal and move it for protecting from early phase after injury. In addition, previous studies have shown that controlling abnormal joint movement in a completely ruptured ACL results in the down-regulation of inflammatory responses [
33] and leads to spontaneous healing [
27]. Furthermore, importance of weight-bearing has been pointed out in previous study [
36]. The results of the present study also showed that an ACL remnant could be maintained by controlling abnormal joint movement. Therefore, controlling abnormal joint movement and weight-bearing may contribute to a spontaneous healing response of the ACL. Furthermore, in the case of ACL reconstruction, the remnant is a very important factor. Previous studies found that the preservation of the remnant tissue improved revascularization and remodeling of the graft and enhanced the biomechanical properties of the graft [
37‐
41]. Consequently, controlling abnormal joint movement and weight-bearing after ACL injury is important not only for conservative therapy but also for patients who undergo ACL reconstruction.
Recent studies have focused on inflammation of the intra-articular tissues after ACL injury [
17,
31]. The coordinated expression of MMP-13 in intra-articular tissues and its accumulation in the synovial fluid may lead to excessive matrix degeneration [
31]. Moreover, increased MMP-13 expression has been implicated in osteoarthritis and rheumatoid arthritis [
42]. Previous studies have also reported that TIMP-1 specifically inhibits MMP-13 [
43,
44], and the MMP-13/TIMP-1 ratio is very important for tissue structure. In normal tissues, the stoichiometric MMP-13/TIMP-1 ratio is 1:1 [
32]. An increase in MMP-13 is associated with the progression of tissue destruction, and an increase of TIMP-1 is associated with the progression of tissue fibrosis [
45]. Compared to the IN and SO groups, the ACL-T and CAM groups showed significantly higher expression levels of MMP-13 and TIMP-1 mRNA and a higher MMP-13/TIMP-1 ratio in all intra-articular tissues. Furthermore, the ACL-T group showed significantly higher MMP-13 mRNA expression compared with the CAM group. Thus, the results suggest that controlling abnormal joint movement after ACL injury inhibited MMP-13 mRNA expression in the intra-articular tissues. Tang et al. reported that MMP-13 mRNA is expressed in the ACL and the intra-articular tissues during the acute phase of ACL injury [
31]. The present study similarly showed that the MMP-13 mRNA expression levels were significantly higher in the intra-articular tissues after the acute phase of ACL injury. The ACL is located within the articular capsule, and after injury, the ACL is exposed to synovial fluid containing inflammatory substances that inhibit healing [
46]. Previous studies have also reported that the expression of MMP-13 mRNA significantly increases in the cartilage and synovium after ACL injury [
31,
47,
48]. Therefore, it is thought that MMP-13 is expressed cooperatively by other intra-articular tissues, accumulates in the synovial fluid, and may promote ACL degeneration after injury. Accordingly, inhibiting the expression of MMP-13 in the intra-articular tissues is considered an important factor in inhibiting the degeneration of articular tissue.
The CAM group showed significantly lower MMP-13 mRNA expression in the intra-articular tissues than did the ACL-T group. The differences in these groups was the use suppression of abnormal movement of the tibia relative to the femur after ACL injury affected our results. Moreover, we showed that the mRNA expression levels of TIMP-1, an inhibitor of MMP-13, increase as the levels of MMP-13 mRNA increase, thereby inhibiting the activity of MMP-13 [
49]. Because the expression of TIMP-1 mRNA in the intra-articular tissues was not significantly different between the ACL-T and CAM groups, the ACL-T group showed a significantly higher MMP-13/TIMP-1 ratio than the CAM group on day 5. Thus, MMP-13 and TIMP-1 were not balanced in the ACL-T group. Moreover, a previous study also indicated that degeneration, such as osseous tissue deterioration, results from the increased expression of MMP relative to that of TIMP [
50]. Therefore, more tissue degeneration was assumed to have occurred in the ACL-T group than in the CAM group based on the dynamics of MMP-13 and TIMP-1.
Previous studies have reported that meniscus injury can occur due to mechanical stress from abnormal movement of the tibia after ACL injury [
2,
50]. Allen et al. reported increases in the front drawer of the tibia and the amount of the load response to the medial meniscus after ACL injury [
51]. Levy et al. reported that the medial meniscus contributes to the braking of the tibial front drawer after ACL injury [
52]. Therefore, mechanical stress to the medial meniscus increases after ACL injury. The lateral meniscus is an important component in the braking of the tibial front drawer. Musahl et al. reported that the amount of forward displacement significantly increased compared to the amount of displacement prior to excision of the lateral meniscus [
53]. Therefore, both the medial meniscus and the lateral meniscus have the ability to brake the tibial front drawer. However, the medial and lateral menisci repeatedly receive mechanical stress from abnormal joint movement after ACL injury. Degeneration of the medial and lateral menisci occurs after an ACL injury due to this repeated secondary mechanical stress. In the present study, the MMP-13 mRNA expression of the medial meniscus and lateral meniscus was higher in the ACL-T group than in the CAM group due to this secondary mechanical stress.
The ACL is responsible for braking the forward movement of the tibia relative to the femur and for tibial rotation. After ACL injury, this braking performance deteriorates, and the loss of this braking capacity in ACL injury patients causes repeated damage to the articular cartilage and meniscus. A previous study reported that more than half of patients with an ACL injury suffer from secondary knee OA [
54]. The results of the present study suggested that the expression of MMP-13 mRNA in the acute phase of ACL injury was inhibited by controlling abnormal joint motion. This finding indicates a possible prevention strategy for joint degeneration after ACL injury. A previous study reported that the occurrence of additional knee injuries increased over time after ACL injury, and the risk of additional meniscus injuries increased substantially 6 months after ACL injury [
55]. ACL reconstruction surgery is therefore recommended within 6 months after injury, but our findings suggest that degeneration of the joint has already occurred during the acute phase of ACL injury. Consequently, joint control during the acute phase is very important for patients with reconstruction therapy after ACL injury.
The present study has several limitations. First, the investigation was a small animal study, which limits the generalizability of the results. The anatomical components of humans and rats are similar; however, the joint function of the knee and weight-bearing conditions are different. These differences affect the joint kinematics of the knee after ACL injury. Thus, the effect of controlled abnormal joint movement in the early phase of complete ACL rupture may differ between humans and rats. Furthermore, although we already performed the same surgical procedure in rabbit ACL, we cannot confirm for healing ACL in the rabbit. We considered that the difference in walking style between rabbits and rats is affecting. Although rats walk for four legs, walk alternately like humans. On the other hands, rabbit moves forward by kicking the ground with both legs. Therefore, it is necessary to select animals with four leg walking (e.g., pig and/or dog) in future research. Second, we studied the healing process of a completely injured ACL only during the early phase (i.e., until post-injury day 5). Third, this study was performed a small sample. Therefore, statistical differences could not be considered in histologic examination. In the future, it is necessary to quantitatively present the narrowing of the ACL stump by increasing the number of samples. Fourth, in the molecular biology evaluation of the present study, we examined only MMP-13 and TIMP-1. Research has shown that inflammatory factors such as cytokines (e.g., interleukin (IL)-6 and IL-8) are present at elevated levels in synovial fluid during the acute phase of ACL injury [
28]. Therefore, it will be ideal to evaluate inflammatory factors in addition to MMPs and 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 inflammatory factors, and another method of ACL rupture are needed to clearly understand the mechanism of spontaneous ACL healing.