Quantitative correlation of mechanoreceptors in tibial remnant of ruptured human anterior cruciate ligament with duration of injury and its significance: an immunohistochemistry-based observational study

Proprioception is a specialized sensory modality encompassing the movement of the joint and its position in space. Presence of mechanoreceptors in the anterior cruciate ligament (ACL) has been well documented in the past by various authors [1‐6]. Thus, the ACL functions as a sensory organ, providing proprioception and stabilizing muscular reflexes while walking. ACL deficiency leads to instability, thus disabling the daily life of an individual [7‐9]. Reconstruction of the ACL does not always yield expected outcome, suggesting that successful reconstruction depends on not only the ultimate strength of the graft but also recovery of proprioception [10‐13]. A few authors have tried to quantify the proprioceptive potential of the injured ACL stump. Denti et al. [14] analyzed 20 injured human ACL stumps using gold chloride stain and found that normal mechanoreceptors persisted for 3 months after injury. After that, there was a gradual decrease in the number of mechanoreceptors (one after 9 months and none after 1 year). However, Dhillon et al. [15] and Georgoulis et al. [16] reported persistence of mechanoreceptors in the ACL stump as late as 42 months and 3 years postinjury, respectively.

Various authors have demonstrated growth of mechanoreceptors in the reconstructed ACL. Barrack et al. [17] found an increase in the mechanoreceptors in the ACL graft 6 months after surgery as compared with the normal patellar tendon in canines. Denti et al. [14] found mechanoreceptors in bone patellar tendon autograft in sheep knees 3 months after implantation, and also in two lax human knees with failed semitendinosus autografts 9 and 10 years after surgery, leading to the conclusion that the ACL remnant is a possible source of neural reinnervation of the graft.

Shaving off the ACL remnant helps improve visualization of landmarks over the tibia and femur and makes surgery technically simpler [18]. Also, preservation of the remnant has been shown to be a risk factor for development of arthrofibrosis and cyclops lesion of the knee [15, 16]. Thus, preservation of the ACL stump has both advantages and disadvantages. We evaluated the proprioceptive potential of the ACL stump in human knees with complete ACL tear using mononuclear antibodies to neurofilament protein (NFP) and correlated the results with duration of injury.

Patients younger than 50 years of age with symptomatic ACL tear were included in this study. All underwent arthroscopic ACL reconstruction with autologous hamstring graft. History of previous knee surgery, tissue with morphological signs of degeneration or inflammatory disorder, lacerated or crushed stumps which precluded adequate sampling, multiligamentous injury, and failure to obtain consent were the exclusion criteria. The study was approved by the Institutional Review Board and Ethics Committee. Ethical standards according to the Helsinki Declaration of 1964 (and its later amendments) were conformed to. Informed consent was obtained from all patients.

A total of 43 patients with symptomatic ACL tear were enrolled in the study, of whom 38 met our inclusion criteria. Three patients had scarred stumps from which adequate sampling could not be done, one patient was diagnosed as a case of ankylosing spondylitis, and one patient had posterior cruciate ligament (PCL) tear on diagnostic arthroscopy. Intraoperatively, residual ACL tissue was harvested by cutting stumps from as near to the attachment site as possible using arthroscopic scissors. When ACL was adhered to PCL, it was carefully dissected off the PCL then resected.

Of the patients, 36 were male and 2 were female. Mean age was 27 years (range 18–39 years, SD 7.21 years). Mean duration of injury was 22 months (range 0.25–120 months, SD 27.74 months). On arthroscopic evaluation, 22 had medial meniscus tear, 12 had lateral meniscus tear, and 6 had both. In 14 knees, the ACL stump was found adhered to the PCL and was carefully separated before harvesting. In four knees, the ACL stump was adhered to the tibial articular surface posterior to the tibial footprint, and 20 were free. In 17 cases, mechanoreceptors were identified, as summarized in Table 1.

Proprioception of the knee is an important consideration for successful ACL reconstruction. The maximum number of nerve endings reside at the attachment site of the ligament to the bone, which serves as the main tract for proprioceptive feedback [14]. Few studies have focused on the presence of mechanoreceptors in the torn ACL stump [2, 12, 14‐17, 19] or its role as a source of neural innervation of the graft. However, some studies have shown that leaving the ACL stump has its own disadvantages such as formation of cyclops lesion, arthrofibrosis, and difficulty in identifying anatomical landmarks during surgery [15, 16].

Three studies employing gold chloride staining to look for mechanoreceptors have been conducted to date [14, 16, 19]. Denti et al. [14] found that mechanoreceptors were detectable up till 3 months after injury, after which they gradually decreased in number. They did not find any mechanoreceptors in any case with duration of injury of more than 1 year. Adachi et al. [19] performed a similar study and found mechanoreceptors in all examined specimens (with duration of injury ranging from 2 months to 10 years). They concluded that there is no correlation between the number of mechanoreceptors and duration of injury, in contradistinction to the finding of Denti et al. Georgoulis et al. [16] found mechanoreceptors in all cases where a portion of the ACL was attached to the PCL, but none or very few in stumps which were free. The shortcomings of gold chloride staining must be acknowledged. Firstly, there is a lack of standardization of the staining technique [2, 3, 23]. Secondly, nonspecific staining of blood vessels, collagen, and elastic fibrils may occur, resulting in incorrect labeling as mechanoreceptors [20, 23]. Third, mechanoreceptors picked up by this method may be morphologically normal while their significance as far as proprioception is concerned is equivocal [1, 15].

In the study conducted by Sha et al. [21], 60 patients undergoing arthroscopic ACL reconstruction were divided into four groups depending upon duration of injury (group I, no more than 3 months; group II, 3–6 months; group III, 6 months to 1 year; group IV, more than 1 year). No significant difference was found among groups I–IV, however the overall trend was for a decrease in the number of mechanoreceptors with the passage of time. Creating similar timeframes for our patients, Fig. 3 shows graphically the difference between the results of the two studies. Although our study shows a trend opposite to that of Sha et al., the increase is statistically insignificant.

Lee et al. [2] used the same immunohistochemical method as applied here, finding 17 mechanoreceptors in 12 out of 36 specimens compared with 19 mechanoreceptors in a control group of 2 specimens. Five of them had necrotized morphology with weak staining and loose form, which was often found in degenerative joint. They speculated that the receptors probably degenerated after injury. The number of receptors was not presented versus duration of injury by Lee et al. In our study, we did not find any mechanoreceptors with necrotic morphology, and 17 out of 38 specimens were positive for mechanoreceptors on the scale of varying time frame.

Dhillon et al. [15] conducted a similar study, where the reactivity to NFP monoclonal antibody for mechanoreceptors in ruptured ACLs was 52.4%, quite similar to the value of 44.7% found in the present study. In contrast to our results, they found a statistically significant relationship between shorter duration of injury and higher proprioceptive potential of injured ACL stump.

Gao et al. [22] found a significant decrease in the number of mechanoreceptors in the ACL stump with increasing duration of injury, which is different from our study. However, they used S-100 instead of NFP staining.

The inconsistency between our and previous studies might be due to variations in specimen collection, sectioning, and processing. First of all, complete harvesting of the specimen was done as close to the attachment as possible in our study, as opposed to some other studies in which the whole residual stump was not harvested. Secondly, most of the other authors using indirect methods applied the S-100 staining method [1, 15, 21, 22]. This method has been proved to stain free nerve endings significantly, but whether these nerve endings have any proprioceptive function remains to be determined [1]. On the other hand, we used monoclonal antibody to NFP, which stains proprioceptive axon cylinders. The validity of this method has been proved by Bali et al. [1]. This also explains the lesser percentage of positive cases in our study compared with previous studies.

The mechanoreceptor detection rate was highest in cases where the ACL stump was attached to the PCL (10 out of 14 cases; 71.4%), whereas the positivity rate was lower in free stumps (5 out of 20 cases; 20%) or where the ACL stump was attached to the tibia (2 out of 4 cases; 50%). This may reflect the higher volume of stump harvested from cases with PCL-adherent stump [15].

Treatment delay is a significant concern in developing countries such as ours. One of the issues that still sparks debate is the persistence of viable mechanoreceptors in the ACL stump and its correlation with duration of injury. Studies have proved that there is a decrease in the mechanoreceptors with increase in duration of injury [14, 18], which may adversely affect the surgical outcome. Mechanoreceptors in the ruptured ACL have also been proposed to play a role in restoring joint stability. Additionally, preserving the native ACL stump can accelerate the cellular proliferation and revascularization of the grafted tendon and improve its biomechanical strength.