Histomorphology of the Subregions of the Scapholunate Interosseous Ligament and Its Enthesis

Abstract

Background  The scapholunate interosseous ligament (SLIL) has three subregions: dorsal, proximal, and volar. The SLIL enthesis has not previously been studied despite its important mechanical function in wrist joint biomechanics.

Questions/Purposes  This study aims to compare the histomorphological differences between the SLIL subregions, including at their entheses. Three questions are explored: Do the gross dimensions differ between SLIL subregions? Does the enthesis qualitatively, and its calcified fibrocartilage (CF) quantitatively, differ between (a) SLIL subregions and (b) scaphoid and lunate attachments?

Methods  Twelve fresh-frozen human cadaveric wrists were dissected and the gross dimensions of the SLIL subregions measured. Subregions were histologically processed for morphological and compositional analyses, including quantification of enthesis CF area.

Results  The dorsal subregion was the thickest. The dorsal and volar subregions had fibrocartilaginous entheses, while the proximal subregion was attached to articular cartilage. The dorsal subregion had significantly more CF than the volar subregion. There was no significant difference in the enthesis CF between scaphoid and lunate attachments in the three subregions.

Conclusions  There are significant morphological differences between the SLIL subregions. The dorsal subregion has the largest amount of CF, which is consistent with the greater biomechanical force subjected to this subregion. The similar histomorphology of the ligament at the scaphoid and lunate entheses suggests that similar biomechanical forces are applied to both attachments.

Clinical Relevance  The histomorphological results confirm that the dorsal subregion is the strongest of the three subregions. The results from the entheseal region may have important implications in the study of graft incorporation during SLIL reconstruction.

The scapholunate interosseous ligament (SLIL) has a unique C-shape ¹ that was historically considered as a single ligament consisting of two portions. ² More recent studies have since defined the SLIL as a single unit with three subregions: dorsal, proximal, and volar. ^{1 2 3} The SLIL is the primary stabilizer of the scapholunate articulation. ^{4 5} SLIL injury follows a progression of ligamentous tearing from the volar to the dorsal subregion. ⁶ The ligament typically avulses from the scaphoid and remains attached to the lunate in acute injuries. ^{5 7 8} If left untreated, this ligament usually does not spontaneously heal and the position of the carpus changes resulting in a predictable pattern of arthritis. ^{5 9} The current most common surgical reconstruction technique is a modified Brunelli procedure. ¹⁰ The aim of this procedure is to restore carpal alignment by using part of a flexor carpi radialis tendon graft passed from volar to dorsal through a tunnel in the scaphoid and attaching it to the dorsal aspect of the lunate. ¹⁰ More recently, and without long-term surgical follow-up results, bone-ligament-bone graft procedures have been described ^{9 11} and these typically focused on reconstructing the dorsal subregion due to ease of surgical access. ¹² Evidence suggests that the dorsal is the strongest and most important subregion to maintain scapholunate interval stability. ^{5 13 14} However, a review of the current literature shows reports varying on the biomechanical strength between different subregions. ³

The enthesis is a specialized region where a tendon, ligament, or joint capsule attaches to bone allowing smooth transition of force between soft tissue and bone. ¹⁵ Entheses are classified as either fibrous or fibrocartilaginous according to the type of tissue found at the attachment site. ^{15 16} A fibrocartilaginous enthesis is characterized by four zones of tissue: dense fibrous connective tissue, uncalcified fibrocartilage (UF), calcified fibrocartilage (CF), and bone. ^{15 17} There is a tidemark between the UF and CF zones that acts as the mechanical boundary between soft and hard tissues. ¹⁸ This attachment site has been widely studied in other anatomical areas such as the anterior cruciate ligament of the knee, ^{17 19} but it has not previously been studied in the SLIL.

The quantification of enthesis CF provides information about load and maximum force transmitted through the ligament–bone junction. ^{15 16 17 20} Previous biomechanical studies were not uniform on the tensile strength of different SLIL subregions. ^{3 12 21 22} Histological quantification of CF area may help to elucidate biomechanical functionality between subregions, as functional adaptation of tissue structure to mechanical force adheres to the “form follows function” principle that underpins Wolff's law. ^{20 23} The gross and histological anatomy of the SLIL have been described in previous studies. ^{1 3 13 24 25 26} However, a systematic review by Buijze et al ² reported inconsistencies in the subregional SLIL description.

This study aims to compare subregional macroscopic and microscopic morphology, including at their entheses, to resolve discrepancies in previously reported studies and better understand the prioritization of any subregion in reconstruction after SLIL injury. A further aim is to compare the entheses of both the scaphoid and lunate to establish differences that may inform the relative frequency of injury between the two bones and to better understand how graft material, used for ligament reconstruction, needs to be incorporated in the bone–ligament interface. The questions explored are: Do the gross dimensions differ between the SLIL subregions? Does the enthesis qualitatively, and its CF quantitatively, differ between (a) SLIL subregions and (b) scaphoid and lunate?

Materials and Methods

Study Design

A total of 12 wrists from 10 fresh-frozen cadavers (age range: 61–87 years; mean 75.8 years; six males, four females) were dissected. All specimens were donated to the University of Edinburgh Medical School body donation program, under the regulations of the Human Tissue (Scotland) Act 2006. The 12 dissected wrists were made up of a left and right wrist from two cadaveric donors and either the left or right wrist from the remaining eight donors. Upon dissection, the SLIL of three wrists showed signs of degeneration and detachment from the carpal bones and were excluded from the study. The remaining nine wrists (all from different cadaveric donors) were taken forward for further analysis.

All wrists specimens were dissected through a longitudinal skin incision, with a standard dorsal approach to expose the scaphoid and lunate bones with the intervening SLIL ( Fig. 1 ). A section through the scaphoid and through the lunate bones with the SLIL intact between them was removed ( Fig. 2 ). Once samples were dissected, they were immediately fixed in 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, Missouri) at 4°C for 48 hours.

Fig. 1.

Dissection of scaphoid and lunate bones with scapholunate interosseous ligament (SLIL). Right wrist in hyperflexion.

Fig. 2.

Dissection of different subregions of the scapholunate interosseous ligament (SLIL). Right wrist in hyperflexion. ( A ) Using an 8 mm osteotome and hammer, the scaphoid and lunate bones were sectioned ∼8 mm from the SLIL attachment to the bones. ( B ) Arrow showing remaining scaphoid and lunate with proximal subregion of SLIL (pSLIL) in situ after the dorsal subregion was removed.

The samples were next decalcified in “Decalcifying Solution-Lite” (Sigma-Aldrich St Louis, Missouri), for 14 to 72 hours, dependent on the size of specimen. The samples were then dehydrated through a series of ethanol concentrations using a VIP E300 tissue processor (Sakura Tissue-Tek, Japan) and mounted in paraffin wax blocks. Ten micrometer sections were cut using a Leica RM 2245 microtome (Leica Microsystems Ltd, UK) parallel with the ligament length, including both bone insertions, at ∼50% through each SLIL subregion ( Fig. 3 ). Multiple sections were mounted onto glass slides and stained with 0.1% toluidine blue (Sigma-Aldrich St Louis, Missouri). The stained slides were scanned using a NanoZoomer-XR C12000 digital slide scanner (Hamamatsu Photonics, Japan) to obtain high-resolution images of the sections for analysis of the SLIL entheses.

Fig. 3.

(Color online) Dimensions of gross measurements taken at each scapholunate interosseous ligament (SLIL) subregion in a right wrist. Green arrows (perpendicular to each subregion), representing direction of sectioning using the microtome. ⊗ represents articulation with lunate bone. ⊙ represents articulation with scaphoid bone. L, length; T, thickness; W, width.

Outcome Measures

On first exposure of the SLIL in dissection, the gross measurements of each SLIL subregion were taken using a digital Vernier caliper, capable of registering to 0.01mm accuracy. Three dimensions were measured: ligament thickness, width, and length ( Fig. 3 ).

For qualitative and quantitative histological study, scanned images of stained sections were analyzed using ImageJ image analysis software (National Institute of Health). Three sections per subregion per wrist were analyzed, and quantitative measurements averaged per subregion. The tissue composition of the ligaments and entheses was observed. In addition, the enthesis profile, defined as the shape of the tidemark at the ligament–bone junction, was categorized as either straight, concave, convex, mixed (concave + convex) or complex (multiple concave + convex profiles). Four quantitative measurements were determined, adapted from a method by Beaulieu et al ¹⁷ —linear enthesis length, segmented enthesis length, CF cross-sectional area, and CF relative area. The linear enthesis length was defined as the linear distance between the edges of the enthesis at the hard–soft tissue junction (enthesis tidemark) ( Fig. 4A–C ). The segmented enthesis length measurement accounted for the curvature of the enthesis by plotting nine points at equidistant intervals (12.5% increments) along the entire linear length and then extrapolating the points perpendicularly to the upper edge of the tidemark ( Fig. 4D ). The enthesis distance was then measured from start to finish between these segmented points.

Fig. 4.

(Color online) Quantification of calcified fibrocartilage cross-sectional area and enthesis length. All images are from the same section. ( A ) Figure showing the scapholunate interosseous ligament (SLIL) attachment to the lunate and scaphoid bones. Enthesis straight length (black line) with arrow on the right marking the beginning of the enthesis, while arrow on the left marking the furthest end of the enthesis, at the tidemark (purple line). ( B ) Magnified image of A, with line and arrow showing the beginning of the enthesis at the scaphoid attachment. ( C ) Magnified image of A, with line and arrow demarcating the transition between the end of the calcified fibrocartilage above and articular cartilage below at the scaphoid attachment. ( D ) Magnified image of A at the scaphoid attachment, showing an example of calcified fibrocartilage cross-sectional area (area enclosed by purple and red lines), linear (black line), and segmented (yellow line) enthesis lengths. The calcified fibrocartilage cross-sectional area lies between the tidemark (purple line) and the calcified cartilage–cortical bone junction (red line). The segmented length is formed by extrapolating the short green lines on the linear enthesis length (black line) to the tidemark. CB, cortical bone; CF, calcified fibrocartilage; F, dense fibrous connective tissue; UF, uncalcified fibrocartilage.

The CF cross-sectional area was defined by the area between the tidemark and the CF–cortical bone junction ( Fig. 4D ). In entheses with multiple tidemarks, the tidemark furthest from the CF–cortical bone junction was selected. The CF relative area was calculated by dividing the CF cross-sectional area by the segmented enthesis length. This method was different from that utilized by Beaulieu et al, ¹⁷ which divided the CF area by linear enthesis length, to control for the curvature of the SLIL entheses. The segmented length was chosen as it was more representative of the curved enthesis profile.

Statistical Analysis

Statistical tests were performed using SPSS Version 24.0 (IBM Corporation) on confirmed normally-distributed data using the Shapiro–Wilk normality test. Repeated measures analysis of variance with a Bonferroni post-hoc analysis was used to compare the gross dimension measurements for each SLIL subregion. Paired t -tests were used to compare the CF area between dorsal and volar SLIL subregions for each bone. Similarly, paired t -tests were used to compare between scaphoid and lunate attachment for each subregion. Statistical significance was defined as p  < 0.05.

Results

From the gross measurements taken ( Fig. 3 ), the SLIL dorsal subregion was significantly thicker than the other subregions ( Fig. 5 ). There were no other significant differences between subregions within other dimensions measured.

Fig. 5.

Differences between the gross measurements of scapholunate interosseous ligament (SLIL) subregions. Mean values with error bars indicating ± one standard error of mean. ** =  p  < 0.01.

From our histological study, the overall composition of the SLIL was different between the three subregions. The dorsal and volar subregions were made up of fibrous connective tissue but the fibrous tissue density was much higher in the dorsal subregion ( Fig. 6A ). The volar subregion had more loosely connected fibrous tissue ( Fig. 6C ). The proximal subregion consisted mainly of fibrocartilage connective tissue ( Fig. 6B ). The dorsal and volar subregions at both the scaphoid and lunate attachments were fibrocartilaginous entheses, whereas the proximal subregion inserted into cortical bone via articular cartilage ( Fig. 7 ). The tidemark of the enthesis was continuous across the adjacent articular cartilage at either end of the enthesis in the dorsal and volar subregions. Both the dorsal and proximal subregions had a mostly convex enthesis profile (Dorsal—50% convex, 27.8% mixed and 22.2% straight; Proximal—81.3% convex, 12.5% mixed and 6.2% straight). About 62.5% of the volar subregion had a complex enthesis profile, 25% were mixed, the rest equally distributed between straight and convex. None of the subregions had a solely concave enthesis profile.

Fig. 6.

Scapholunate interosseous ligament (SLIL) subregions from the same wrist specimen stained with toluidine blue. ( A ) Fibrous connective tissue of the dorsal subregion. ( B ) Proximal subregion showing distinct purple (darker) staining of cartilage proteoglycans and presence of chondrocytes in the ligament region. ( C ) Fibrous connective tissue of the volar subregion. dSLIL, dorsal SLIL; pSLIL, proximal SLIL; vSLIL, volar SLIL.

Fig. 7.

Magnified scaphoid enthesis of each scapholunate interosseous ligament (SLIL) subregion (from Fig. 6 ). Arrows indicate enthesis tidemark. ( A ) Dorsal subregion. ( B ) Proximal subregion. ( C ) Volar subregion. AC, articular cartilage; CB, cortical bone; CF, calcified fibrocartilage; F, fibrous connective tissue; L, ligament, UF, uncalcified fibrocartilage.

From the measure of enthesis CF, the dorsal subregion had significantly more CF than the volar subregion. The dorsal subregion was 1.97 times thicker than the volar subregion (value calculated by comparing mean CF relative area between the two subregions at both scaphoid and lunate attachments). There were no significant differences in enthesis CF area between the scaphoid and lunate in either subregion ( Fig. 8 ). Analysis of the linear and segmented enthesis lengths showed no significant differences between the subregions or between the scaphoid and lunate.

Fig. 8.

Quantification of calcified fibrocartilage (CF) area. ( A ) Mean cross-sectional area of enthesis CF. ( B ) Mean relative area of enthesis CF. Relative area was calculated by dividing CF cross-sectional area by the segmented enthesis length. Error bars indicating ± one standard error of mean. * =  p  < 0.05 and ** =  p  < 0.01.

Discussion

The enthesis is the region where a tendon, ligament, or joint capsule attaches to bone. ¹⁵ It has been widely studied in other anatomical areas such as the anterior cruciate ligament of the knee, ^{17 19} but there is no existing literature on the enthesis of the SLIL. This ligament is divided into three subregions: dorsal, proximal, and volar. ¹ Surgical reconstruction following SLIL injury commonly employs a modification of the Brunelli technique, recreating the scapholunate positioning, with the tendon graft passing through the scaphoid, out dorsally and attaching onto the lunate in the dorsal subregion. ¹³ When bone–ligament–bone grafts are used, the graft is placed through a more accessible dorsal wrist arthrotomy. ^{7 12} Previous evidence suggested that this is the strongest and most important subregion to maintain scapholunate interval stability. ^{3 27} However, a recent review reported inconsistencies in the biomechanical strength and functionality between different SLIL subregions. ³

This study acknowledges several limitations. Sample size was limited and within a regional population. Differences described were, however, clear and seen in comparison of all wrist specimens, suggesting a population trait. The age range of cadaveric specimens was between 61 and 87 years old. The enthesis is known to undergo age-related degeneration, including an increase in thickness of CF ^{17 28} however, this seems very unlikely to potentially affect one bone or subregion preferentially. ¹⁷ We would postulate that the comparative differences found will still be present in a younger population. Analysis of the SLIL, a three-dimensional structure, was performed using two-dimensional images. Every effort was made to achieve reliable and consistent region sampling of sections at the middle 50% of each subregion, in a plane perpendicular to the articular surface of the carpal bones ( Fig. 3 ).

The thickness measurement was the main gross morphological difference between the SLIL subregions. Our results showed that the dorsal subregion was on average twice as thick as the proximal or volar subregion. A systematic review by Buijze et al ² highlighted controversies on gross measurements of SLIL subregions. These authors reported average gross measurements based on their review of four different studies. ^{1 25 26 29} Our results on ligament thickness and length mirrored the overall measurements reported in this systematic review. ² This verifies that the dorsal subregion is indeed much thicker than the other two subregions, while there is no significant difference in ligament length between subregions. However, there were discrepancies between our width measurements and the measurements previously reported. Our review of those studies revealed that the authors did not specify their technique of ligament measurements. This, coupled with the difficulty in measuring a “linear” width for a “curved” C-shaped SLIL, could be possible explanations for these discrepancies.

The dorsal and volar subregions were mainly collagenous, while the proximal subregion was largely fibrocartilaginous, in agreement with findings from previous studies. ^{1 24} Hence, it is natural to postulate a fundamental difference between the proximal and dorsal/volar subregions in terms of function due to their different principal tissue types. This is consistent with the clinical finding of perforations seen solely in the proximal SLIL subregion at wrist arthroscopy, not causing scapholunate instability. From our results, the entheses at the dorsal and volar subregions could be classified as fibrocartilaginous entheses with four zones of tissue found at the attachment site: dense fibrous connective tissue, UF, CF, and cortical bone. The fibrocartilage in the proximal subregion inserted into cortical bone via articular cartilage and the four distinct tissue zones were not present. This finding suggests a key difference in biomechanical functionality between the proximal and the dorsal and volar subregions as the quantity of each tissue type found is characteristic of mechanical loading at the enthesis. ^{17 20}

The quantification of fibrocartilaginous enthesis CF informs about load and maximum force transmitting through the attachment site. ^{17 19 20 30} We considered relative quantitative enthesis comparison between the dorsal and volar subregions possible as they had fibrocartilaginous entheses, while the proximal subregion did not and was thus excluded. Previous biomechanical studies focused mainly on the dorsal subregion. ³ The few biomechanical studies that conducted comparisons between the three subregions found varying results ³ : most agreed that the dorsal subregion was the strongest, ^{5 7 11 12 24 26} Nikolopoulos et al ²¹ reported approximately equal strength between the dorsal and volar subregions, while Logan et al ²² reported that the volar subregion was the strongest. However, the general consensus was that the proximal subregion is the only subregion that is not considered in SLIL reconstruction due to its smallest mechanical contribution. ^{3 12 24} A greater CF area is a measure of greater mechanical force at the enthesis. ^{18 30 31} Our study showed that the dorsal subregion had a much greater CF area compared with the volar subregion. This suggests that the dorsal subregion takes the greatest load and therefore is the most important subregion in supporting the scapholunate interval. This is supported by previous biomechanical studies which agreed that the dorsal subregion is subjected to the greatest biomechanical force. ^{5 7 11 12 24 26} Since the relative CF area, which controlled for different insertion lengths of the enthesis, showed a similar doubling of CF area in the dorsal compared with volar subregion, the dorsal CF area could also be described as twice as thick, further emphasizing the greater load transmitted through this subregion. Clinical studies have shown that avulsion tends to occur from the scaphoid attachment in acute injuries. ^{5 7 8} Our study found no significant difference in CF area between the scaphoid and lunate in all three subregions. This suggests that other factors, including pattern of force transfer during injury, may have more influence on SLIL avulsion sites.

In conclusion, there are significant differences in macroscopic and microscopic morphology between the SLIL subregions. Our results, coupled with analysis of previous literature, confirm that we should consider the SLIL as three distinct morphological subregions, for reconstruction and function. ⁶ We found the dorsal subregion to be the thickest and contain the greatest proportion of CF, consistent with its role as the most important SLIL subregion for biomechanical strength and to maintain scapholunate interval stability. However, we found no significant difference in proportion of CF between the scaphoid and lunate attachments of the ligament, suggesting that this is not the main factor in determining the site of ligament avulsion. Further studies can be performed to examine other factors influencing ligament avulsion from either carpal bone in acute injuries. This study also provides insight into the bone–ligament interface and tissue ingrowth necessary for the successful incorporation of neoligament graft material.

Funding Statement

Funding This research received grants from Hand and Wrist Medical Research Foundation, a charitable body registered in Melbourne, Australia and grants from Mr Hamilton’s Research and Development Fund, St John’s Hospital, Edinburgh, during the conduct of the study.