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Histological, radiological and clinical analysis of the supraspinatus tendon and muscle in rotator cuff tears

BMC Musculoskeletal Disorders volume 24, Article number: 127 (2023) Cite this article

Abstract

Background

Macroscopic alterations of the affected rotator cuff (RC) are undoubtedly linked to microscopic changes, but they may underestimate the actual degree of the disease. Moreover, it remains unclear whether preoperative structural RC changes may alter clinical outcomes.

Methods

Supraspinatus tendon and muscle samples were collected from 47 patients undergoing RC surgery. Tendons were evaluated histologically according to the Bonar score; fatty infiltration and muscle atrophy were quantified using a software for biomedical image analysis (ImageJ) in percentage of area affected in the observed muscle section. Preoperative shoulder ROM and pain were evaluated. Radiological muscle atrophy was evaluated with the Tangent Sign and Occupation Ratio; fatty infiltration was assessed according to the Goutallier classification. Correlations between histological, radiological and clinical outcomes were assessed. Statistics were performed using the Spearman correlation coefficient. Intraobserver and interobserver agreement was calculated.

Results

Histopathologic fatty infiltration (r = 0.007, p = 0.962), muscle atrophy (r = 0.003, p = 0.984) and the total Bonar score (r = 0.157, p = 0.292) were not correlated to preoperative shoulder pain. Muscle atrophy showed a significant but weak negative correlation with the preoperative movement of abduction (r = -0.344, p = 0.018). A significant but weak positive correlation was found between muscle atrophy and the total Bonar score (r = 0.352, p = 0.015). No correlation between histological and radiological evaluation was found for both fatty infiltration (r = 0.099, p = 0.510) and muscle atrophy (Tangent Sign: r = -0.223, p = 0.131; Occupation Ratio: r = -0.148, p = 0.319). Our histological evaluation showed a modal value of 3 (out of 3) for fatty infiltration and an equal modal value of 2 and 3 (out of 3) for muscle atrophy. In contrast, the modal value of the Goutallier score was 1 (out of 4) and 28 patients out of 47 showed a negative Tangent sign. At histology, intraobserver agreement ranged from 0.59 to 0.81 and interobserver agreement from 0.57 to 0.64. On the MRI intraobserver agreement ranged from 0.57 to 0.71 and interobserver agreement ranged from 0.53 to 0.65.

Conclusions

Microscopic muscle atrophy appeared to negatively correlate with the movement of abduction leading to functional impairment. Shoulder pain did not show any relationship with microscopic changes. Radiological evaluation of the supraspinatus muscle alterations seemed to underestimate the degree of the same abnormalities evaluated at histology.

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Introduction

Rotator cuff (RC) disease represents a common source of shoulder pain and dysfunction in adults, although not all patients are symptomatic [1]. The pathogenesis and the natural history of RC tendinopathy is still under debate. It is considered a multifactorial disease [2], with intrinsic (age-related degeneration, genetic predisposition) and extrinsic (micro and macro-traumas) cofactors that contribute to weaken tendons until their rupture [3, 4]. Smoking, hypercholesterolemia and genetics are all proven risk factors for the development of RC tears, influencing the age-related tendon degeneration process [5, 6]. Recently, a gender predisposition for the development of the disease has been theorized [7]. Previous studies have found that, in the early stages of the disease, the supraspinatus tendon may already show the typical histopathologic changes of RC tendinopathy although it appears macroscopically intact [3]. These microscopic tendon alterations promote the progression of pre-existing lesions [8] and predispose the RC to rupture in presence of repeated external traumas [9]. Degenerative changes have been commonly found in many cadaver supraspinatus with no history of shoulder pain [10]. Biochemical changes in tendon matrix are known to predispose a proportion of individuals to chronic rotator cuff tendinitis [11]. In accordance, Hashimoto et al. [12] showed that pathologic tendon changes predate the rupture. The supraspinatus muscle atrophy and fatty infiltration are classic histomorphological changes that develop within muscles after RC tendon tears [8, 13]. They do not improve even after a successful RC repair and the best possibility is the preservation of the preoperative status [8, 13]. Therefore, these findings influence the effectiveness of surgical treatment in terms of functional and symptomatic recovery [14]. Fatty infiltration determines muscle weakness and increases surgical failure rates [15], whereas muscle atrophy is responsible for a reduction of contractile strength [16]. The development of these alterations is probably due to adaptive mechanisms secondary to tendinopathy and they are thought to contribute to the progression of RC lesions [8]. This study aimed to perform a multiparametric analysis of the RC disease, looking for potential relationships between clinical, histological and radiological evaluations. We hypothesize that macroscopic (clinical and radiological) evaluation of the supraspinatus alterations may underestimate the actual (microscopic) degree of shoulder disease. Moreover, it remains unclear whether preoperative structural RC changes may alter clinical outcomes.

Materials and methods

The Ethics committee of our University approved the present study. Informed consent was obtained from all participants.

Patients

The study enrolled 77 patients representing a consecutive series of patients treated at the orthopedic department of our institution from November 2017 to May 2019. 9 patients refused to participate to the present study and 21 patients met the exclusion criteria. Overall, we collected full data of 47 patients. All the patients included in the present study undergoing surgical repair had a diagnosis of RC tear. All patients underwent preoperative shoulder magnetic resonance imaging (MRI) scan in the affected side, performed with a 1.5-T unit. Full-thickness rotator cuff tears were measured on the sagittal (at the tuberosities) and coronal images and classified into the numbers of tendons torn by the Senior Author: small tears (< 1 cm of tear and < 1 tendon involved, also called punctiform tear); medium (1–3 cm of tear and 1 tendon involved); large (3–5 cm of tear and 2 tendons involved); massive (> 5 cm of tear and > 2 tendons involved) [17]. Partial-thickness rotator cuff tears were graded by the Senior Author in a binary fashion as either grade 1 (less than 50% torn) or grade 2 (equal or more than 50% torn) and according to the side torn (either articular or bursal) [18, 19]. Conservative management, including nonsteroidal anti-inflammatory drugs, physiotherapy, and rest, failed in all patients, and they continued to experience pain and functional limitation in the affected shoulder. All patients fulfilled the following inclusion criteria: (1) positive RC pathology signs on preoperative examination (at least 1 among Jobe test, Napoleon test, lift-off test, and Patte test) [20], (2) no episodes of shoulder instability, (3) no radiographic evidence of fracture of the glenoid or the tuberosities, (4) MRI evidence of RC tear, (5) RC tear of 1 or more tendons at arthroscopic examination, and (6) no traumatic lesion (lesions involving more than 1 cm of the labrum) of the glenoid labrum or of the capsule at arthroscopic examination. Exclusion criteria were: (1) injections of corticosteroids in the affected shoulder, (2) previous surgical treatment in the shoulder; (3) arthritis of the acromioclavicular or the glenohumeral joint; (4) history of trauma on the affected shoulder.

Samples collection

At arthroscopy, biopsies (about 4 × 4 mm in size) of the supraspinatus tendon (Fig. 1A) and muscle (Fig. 1B) were collected by means of arthroscopic instruments. Tendon specimens were harvested within the arthroscopically intact middle portion of the tendon between the lateral edge of the tendon tear and the muscle–tendon junction. Muscle specimens were collected at the muscle–tendon junction.

Fig. 1
figure 1

Withdrawal of the supraspinatus tendon and muscle bioptic samples during arthroscopic rotator cuff repair by means of arthroscopic instruments. A Withdrawal of a supraspinatus tendon specimen. B Withdrawal of a supraspinatus muscle specimen

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Histopathology and immunohistochemistry

After removing, tissue samples were promptly fixed in buffered formalin 10% at room temperature for 24 h. They were rinsed in phosphate buffered saline (PBS, pH 7,4), dehydrated in an ascending series of alcohol and embedded in paraffin via xylene. Then, 3–5 μm serial sections were cut and processed for Haematoxylin/Eosin, Masson–Goldner Trichrome, Alcian Blu and immunohistochemical stainings. Immunohistochemical analysis was performed using the indirect technique [16, 21]. Sections were deparaffinized and endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide for 5 min at room temperature. The following antibodies were used: mouse monoclonal antibody anti-CD34 (1:100 titre clone QBEnd/10 CM084B; Biocare – Italy) for the evaluation of the microvascular density. After washing with Tris-Buffered saline (TBS), sections were incubated with their primary antibody: CD34 for 2 h at RT. Preparations were then washed three times with PBS, and incubated with their secondary antibody following the kit protocol Dako EnVision Dual Link System-HRP: antimouse (EnVision FLEX LINKER mouse) for 15 min. Sections were then washed three times with TBS and finally incubated in diaminobenzidine (DAB, Dako), for 5 min, followed by haematoxylin counterstaining. Semiquantitative evaluation of immunoreactivity was performed at × 40 magnification in 10 microscopic fields randomly chosen by means of a scientific digital camera (SPOT Insight; Diagnostic Instrument, Inc., Sterling Heights, MI, USA) connected to an Olympus BX-51 light microscope (Olympus, Tokyo, Japan) and elaborated with an Image Analysis System (Delta Sistemi). Negative control slides processed without primary antibodies were included for each staining. Fatty infiltration and muscle atrophy of the supraspinatus muscle were quantified using a software for biomedical image analysis (ImageJ) in percentage of area affected with respect to the whole area of the observed section. For each specimen, fatty infiltration was classified into three grades: grade 1 (< 10%); grade 2 (10–20%); grade 3 (> 20%) of adipose tissue area. Similarly, muscle atrophy was evaluated on sections stained with Masson–Goldner Trichrome, considering three grades of alteration: grade 1 (< 10%); grade 2 (10–20%); grade 3 (> 20%). Semiquantitative analysis of tendinopathy was performed at × 4, × 10, × 20 magnification according to the Bonar score [22, 23]. It includes four parameters: tenocytes, ground substance, collagen, vascularity. Each parameter is scored with a four-point scoring system, in which 0 indicates healthy tissue and 3 highly degenerated tissue. The total Bonar score is comprised between 0 (normal tendon) and 12 (most severe abnormality detectable). For each muscle and tendon specimen, 3 slides were randomly selected and examined with a light microscope. The identification number on each slide was covered with a removable sticker, and each slide was numbered using randomly generated numbers. After 1 of the authors interpreted all the slides once, the stickers were removed, a new sticker was applied, and the slides were renumbered using a new series of randomly generated numbers. The degree of staining of all the slides was reassessed by the same author (an Orthopaedic and Trauma Surgery PhD Student), and the two results were compared. When there was only a 1-grade difference between the two assessments, the highest result was chosen. If an inconsistency (> 1 grade on the scoring system described below) existed between the 2 results, the slides were reassessed with the help of a board certified pathologist (sixth author). Intraobserver and interobserver agreement was calculated.

Functional assessment

The preoperative range of motion (ROM) of the affected joint was measured. An universal standard goniometer with 1 degree increments was adopted and guidelines for the evaluation of ROMs were followed [24, 25]. Active abduction and external rotation (at 0° and 90° of abduction) of the affected shoulder were recorded preoperatively for each patient. ROM assessment was repeated three times sequentially and arithmetic average was used for statistical analysis. Preoperative shoulder pain evaluation was performed with a visual analogue scale (VAS) pain score (it ranges from 0 cm = “no pain”, to 10 cm = “worst imaginable pain”) [26].

Radiological assessment

A T2-weighted MRI study's most lateral scan, in which the spine is in touch with the scapular body, is used to analyze the tangent sign [27]. The tangent sign is determined by drawing a line connecting the superior edge of the scapula's spine to the superior edge of the coracoid process. The muscle content should cross superior to the tangent line in healthy tissues. The superior border of the muscle must fell below the tangent line when the tangent sign is positive (showing significant atrophy). When the tangent line and the muscular belly of the supraspinatus intersect, the tangent sign is negative [27].

Oblique sagittal PD-weighted MRI scans were used to calculate the occupation ratio. The boundary of the supraspinatus fossa was closely followed as possible and the supraspinatus muscle outer rim was traced at the most lateral portion of the scapula's Y-view appearance in the oblique sagittal plane. The superior limit of the supraspinatus fossa was the distal clavicle. We used a formula to determine the Occupation Ratio as a measure of muscle volume using the technique provided by Thomazeau et al. [28]. Then, using the formula S1 / S2, the occupation ratio was determined. S1 represents the surface area of the supraspinatus muscle, and S2 represents the surface area of the entire supraspinatus fossa. If the ratio (stage 1) is between 1.00 and 0.60, the muscle is either normal or mildly atrophying. Significant atrophy is indicated by values between 0.60 and 0.40 at the stage 2 level. Stage 3 values below 0.40 indicate severe or moderate atrophy [28].

Using the Goutallier classification as modified by Fuchs et al. [29] on T1-weighted turbo spin-echo sequences, all muscles were evaluated at the most lateral scan on the sagittal view, where the spine was in contact with the scapular body [29] Two additional scans were taken into account for the infraspinatus and subscapularis tendons: an inferior scan at the lowest point of the glenohumeral joint and a superior scan at the level of the lateral attachment of the spine. Five stages are used to categorize changes: Stages 0 and 1 indicate no fat, stage 2 indicates there is more muscle than fat, stage 3 indicates there is an equal amount of fat and muscle, and stage 4 indicates there is more fat than muscle [30].

The MRI images were reassessed by the same author (an Orthopaedic and Trauma Surgery PhD Student), and the two results were compared. The author who performed the evaluation was blinded to clinical findings and to patient’s identities. Intraobserver and interobserver agreement was calculated. When there was only a 1-grade difference between the two assessments, the highest result was chosen. If an inconsistency (> 1 grade on the scoring system described below) existed between the 2 results, the slides were reassessed with the help of a board certified orthopaedist (Senior Author).

Statistical analysis

Data are expressed as mean and standard deviation (± SD). Since data showed an abnormal distribution, statistics were performed using the Spearman correlation coefficient. Potential correlations were assessed at histology between tendon and muscle alterations (muscle atrophy and fatty infiltration); between histological muscle atrophy and fatty infiltration themselves; between muscle atrophy evaluated at histology and on the MRI; between fatty infiltration evaluated at histology and on the MRI; between histological tendon alterations and clinical outcomes (VAS pain and ROM); between histological muscle atrophy and clinical outcomes (VAS pain and ROM); between fatty infiltration and clinical outcomes (VAS pain and ROM). Only p values < 0,05 were considered to be statistically significant. Kappa statistics were used to assess the intraobserver and interobserver agreement.

Results

The study assessed 47 patients treated at our institution. The mean age of patients was 62.5 years (± 7.4, range 52–78). The rotator cuff tears were preoperatively classified on the MRI as small (< 1 cm) in 5 patients, medium (1- 3 cm) in 12 patients, large (3–5 cm) in 18 patients, and massive (> 5 cm) in 12 patients. Biopsy samples of the supraspinatus tendon and muscle were collected from 19 women (mean age 62.3 ± 8.1) and 28 men (mean age 62.7 ± 6.8) undergoing surgery for RC tears. Histomorphological evaluation of tendon alterations revealed a mean value of the total Bonar scale of 6.5 ± 1.9 (range from 2 to 10) (Table 1).

Table 1 Summary of Pathologic scores of the Supraspinatus Tendon. The worst scoring result was used for each situation
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Specimens stained with Haematoxylin / Eosin showed abnormalities of tenocyte morphology in terms of nuclear shape and dimension (Fig. 2A). Grade 2 alteration was the modal value. The immunohistochemical evaluation of tendon vascularity showed occasional pathological clusters of capillaries (Fig. 2B). Grade 1 alteration was the modal value. Alcian blu stainings showed ground substance abnormalities in tendon tissue: intrafascicular and then interfascicular deposits of glycosaminoglycans (GAG) disarraying collagen fibers (Fig. 2C). Grade 1 alteration was the modal value. Masson–Goldner Trichrome and Haematoxylin / Eosin stainings showed disorganization of collagen fibre bundles (Fig. 2D). Grade 2 alteration was the modal value. Masson–Goldner Trichrome stainings allowed the evaluation of the supraspinatus muscle atrophy: amounts of interfibrillar connective tissue mainly composed of disorganized collagen fibers were observed in pathologic tissue samples (Fig. 3A). Grades 2 and 3 were the modal values. Haematoxylin/Eosin stainings of the supraspinatus muscle showed intrafascicular and, in severe cases, perifascicular fatty infiltration [15] (Fig. 3B). Grade 3 level of expression was the modal value (Table 2).

Fig. 2
figure 2

Histomorphological analysis of the supraspinatus tendon alterations according to the Bonar Score. A Haematoxylin/Eosin staining showed severe morphological alteration of tenocytes. B CD34 immunohistochemical staining shows tendon vascularization. C Alcian Blu staining highlights ground substance abnormalities. D Haematoxylin/Eosin staining shows disorganization of collagen fibre bundles. Original magnification: × 200 (A), × 100 (B, C), × 150 (D)

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Fig. 3
figure 3

Histomorphological analysis of the supraspinatus muscle alterations. A Masson–Goldner Trichrome staining shows fibrous infiltration, responsible for muscle atrophy. B Haematoxylin/Eosin staining shows fatty infiltration, surrounding the residual muscle fibers. Original magnification: × 100 (A, B)

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Table 2 Summary of Pathologic scores of the Supraspinatus Muscle. The worst scoring result was used for each situation
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Statistical analysis revealed that histopathological fatty infiltration does not correlate with the preoperative VAS shoulder pain score (r = 0.007, p = 0.962) (Table 3).

Table 3 Summary of ROM assessment and VAS Pain Score. ROM assessment was repeated three times and arithmetic average was used for statistical analysis
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Moreover, fatty infiltration did not correlate with preoperative movements of abduction (r = 0.139, p = 0.351), external rotation at 0° of abduction (r = 0.046, p = 0.756), external rotation at 90° of abduction (r = 0.034, p = 0.819). No statistically significant correlation was found between histopathological and radiological fatty infiltration (r = 0.099, p = 0.510) (Table 4).

Table 4 Summary of the population assessed for the Goutallier Score. The worst scoring result was used for each situation
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Histopathological supraspinatus muscle atrophy did not correlate with the VAS shoulder pain score (r = 0.003, p = 0.984). No significant correlation was found between histopathological supraspinatus muscle atrophy and preoperative movements of external rotation at 0° of abduction (r = -0.221, p = 0.135), external rotation at 90° of abduction (r = -0.096, p = 0.522). In contrast, there was a significant but weak negative correlation between histopathological muscle atrophy and the preoperative movement of abduction (r = -0.344, p = 0.018). No significant correlation was found between the expression of histopathological muscle atrophy and radiological muscle atrophy (Tangent sign: r = -0.223, p = 0.131; Occupation Ratio: r = 0.122, p = 0.413) (Table 5). The total Bonar score did not correlate with the VAS shoulder pain score (r = 0.157, p = 0.292). No significant correlation was found between the total Bonar score and preoperative movements of abduction (r = -0.084, p = 0.576), external rotation at 0° of abduction (r = -0.281, p = 0.055), external rotation at 90° of abduction (r = -0.074, p = 0.622). At histology, no significant correlation was found between the supraspinatus muscle atrophy and fatty infiltration (r = -0.263, p = 0.074); no significant correlation was found between fatty infiltration and the total Bonar score (r = 0.095, p = 0.527); a statistically significant but weak positive correlation was found between the supraspinatus muscle atrophy and the total Bonar score (r = 0.352, p = 0.015). Using the kappa statistics, at histology intraobserver agreement ranged from 0.59 to 0.81 and interobserver agreement from 0.57 to 0.64. On the MRI intraobserver agreement ranged from 0.57 to 0.71 and interobserver agreement ranged from 0.53 to 0.65 (Table 6).

Table 5 Summary of the population assessed for the Thomazeau Stage, the Occupation Ratio and the Tangent Sign
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Table 6 Kappa Scores for Each Variable. Kappa Score: 1 indicates a perfect match, and 0 represents no match
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Discussion

This study has shown that the supraspinatus tendon and muscle of patients with RC tendinopathy present profound histopathological changes, that in part justify the clinical shoulder impairment. However, no correlation was found between histological and radiological evaluation of fatty infiltration and muscle atrophy.

Histopathology of the degenerated supraspinatus tendon

In healthy tendons, tenocytes and their precursors constitute about 95% of the cellular elements [31]. Tenocytes are flat, tapered cells sparingly distributed among collagen fibrils [31]. They synthesize collagen and all components of the extracellular matrix network [32, 33]. In our samples, tendons showed alterations of tenocyte morphology (in terms of nuclear shape and dimension). It confirms tenocyte changes in tendinopathy with altered tendon cells resembling chondrocytes [31]. Masson–Goldner Trichrome stainings showed disorganization of collagen fibre bundles. The literature indicates that, in healthy tendons, microscopy reveals a hierarchical arrangement of tightly packed, parallel bundles of collagen fibres [34]. Results of the present study confirm the typical modifications of the collagen fibre bundles that become separate and disorganized in patients with RC tendinopathy [35, 36]. In healthy tendons, stainable ground substance (extracellular matrix) is generally absent [34]. In this study, Alcian Blu stainings revealed ground substance abnormalities: intrafascicular and then interfascicular deposits of GAG disarraying collagen fibers. It is known that areas of altered collagen fibre structure and increased interfibrillar ground substance, which has been shown to consist of hydrophilic GAG, correspond with increased signal on the MRI [37] and hypoechogenic regions on ultrasound investigation [38, 39]. The immunohistochemical evaluation of tendon vascularity showed occasional pathological clusters of capillaries. In general, specimens showed low levels of CD34 expression, with a grade 1 alteration mainly observed. As regards the shoulder, vascular insufficiency has been proposed as one of the potential causes of RC disease [40, 41]. In particular, has been shown that diminished vascular supply is associated with degenerative RC lesions [42, 43]. These results are consistent with previous studies in which blood flow and vascular density in the affected supraspinatus tendon appeared to be significantly lower if compared with the normal tendon [22, 44, 45].

Histopathology of the degenerated supraspinatus muscle

Persistent atrophy of muscle fibers and an accumulation of fat, commonly referred to as fatty infiltration, generally occur in patients with chronic and massive RC tears [30]. The etiology of fatty infiltration and function of the residual rotator cuff musculature have not been well characterized yet [46]. Current surgical treatments are unable to alter or reverse the progression of fatty infiltration and are associated with poor functional outcomes in these patients [47, 48]. Our Haematoxylin / Eosin stainings of the supraspinatus muscle showed high levels of fatty infiltration, with intrafascicular and, in severe cases, perifascicular localization. Moreover, Masson–Goldner Trichrome stainings allowed the evaluation of the supraspinatus muscle atrophy: amounts of interfibrillar connective tissue mainly composed of disorganized collagen fibers were observed in pathologic tissue samples.

Reliability of histopathological and radiological interpretation

In this study, each specimen was assessed twice with the help of a board certified pathologist. Also the radiological evaluations were repeated twice with the help of a board certified orthopaedist. The kappa statistics showed that the intraobserver and interobserver agreement of blinded assessment for the various tendon and muscle scoring systems is acceptable. Whether these methods could be implemented in clinical practice or in research studies is open to discussion [49].

Clinical implications

A clinically relevant finding is that, microscopically, the quantification of degree of tendon alteration confirmed to be strictly linked to the degree of muscle atrophy. This finding remarks the importance of an early diagnosis for RC tendinopathy: the activation of molecular patterns in the adjacent muscle, consequent to tendon tears, is responsible for a progressive and irreversible muscular involution [13, 50]. Despite fatty infiltration and muscle atrophy are classic morphologic changes in chronic RC tears, how these two alterations develop and what are the molecular driving patterns remains unclear [8, 13, 50]. Results of the present study did not show any reciprocal influence of these two abnormalities on muscles. It can be speculated that, although they are both features of the same disease, they may be driven by different pathogenic triggers. Our evidence that tendon alterations were linked to the degree of muscle atrophy but independent from fatty infiltration seems to be in line with this theory. This is in accordance with previous studies where, on patients who had undergone successful RC repair, no relationships were found in the postoperative evolution of muscle atrophy and fatty infiltration [14].

The association of structural characteristics with pain and function in patients with RC tears is still debated [6, 51, 52]. This study reported that microscopic tendon alterations, fatty infiltration and muscle atrophy do not correlate with shoulder pain. Theses results are in line with the current literature: previous studies have shown that morphological tear severity is not associated with patient-reported shoulder pain [6, 51, 53]. In contrast, other authors highlighted that biomechanical, epidemiological and psychological factors may influence shoulder pain patient perception [6, 51, 53].

The supraspinatus compresses, abducts and provides a small external rotation torque to the glenohumeral joint [54]. Studies have demonstrated the contribution of the supraspinatus to abduction [55, 56]. Furthermore, this muscle provides weak external rotation regardless of abduction angle, although it appears to be a more effective external rotator at smaller abduction angles [57]. In the present study, the active preoperative ROM of the shoulder was recorded. Results showed a statistically significant correlation between histopathological muscle atrophy and the preoperative movement of abduction. The correlation is negative, it means that the more muscle atrophy increases, the more the abduction movement is impaired. We have harvested only supraspinatus samples, in patients with rotator cuff tendinopathy. When left untreated, supraspinatus muscle atrophy and fatty infiltration typically progress over time from the moment of tendon detachment [58]. Mechanical detachment of the tendon is the primarily responsible for fatty infiltration and rotator cuff atrophy. Moreover, time from onset of symptoms to diagnosis is associated with progression of these two abnormalities [58]. Muscle atrophy is believed to be irreversible and contributes to poor functional outcomes after tendon repair [8, 13, 50]. It can be assumed that higher levels of muscle atrophy belong to older lesions, with a more heterogeneous impairment of the rotator cuff components. Accordingly, results of the present study have found a negative correlation between supraspinatus muscle atrophy and loss of abduction. These findings suggest a major role of the muscle atrophy, rather than tendon degeneration and fatty infiltration, in impairing the supraspinatus clinical function. Surprisingly, histological and radiological fatty infiltration did not show any significant correlation. The same occurred for muscle atrophy. This result may be in part attributable to the spareness of our population. Our histological evaluation showed a modal value of 3 (out of 3) for fatty infiltration and an equal modal value of 2 and 3 (out of 3) for muscle atrophy. In contrast, the modal value of the Goutallier score was 1 (out of 4) and 28 patients out of 47 showed a negative Tangent sign. These discrepancies in grading the same alterations by means of different techniques suggest that macroscopic (clinical and radiological) evaluation of the shoulder may underestimate the actual degree of disease. These may lead to late diagnosis increasing failure rates [59].

Limitations of the present study

We are fully aware of the limitations of this study. For example, a relatively small sample size did not allow us to perform large-scale statistical analyses. Moreover, we did not have any control group of unaffected supraspinatus muscle and tendon samples. The ideal control should not have any shoulder abnormalities and systemic disease. due to the lack of a control group we were unable to compare our findings with normal healthy tissue. However, for ethical and practical reasons, no alternatives were possible, as it is impossible in our setting to take surgical biopsy specimens from healthy individuals. When interpreting the results of this study, it should be pointed out that we used only 3 staining methods and 1 immunohistochemical analysis. Obviously, more advanced histochemical and immunohistochemical techniques and electron microscopy analysis would allow better estimation of the supraspinatus abnormalities. However, stainings employed in the present study are widely available, cost-effective, and require little technical ability. We harvested muscle and tendon specimens from the supraspinatus myo-tendinous junction: there is still no evidence that the status of the myo-tendinous junction is the same of the muscle or tendon core. Furthermore, in the present study, 2D histology with 2D imaging were correlated: It is known that the muscle status changes over its course from medial to lateral and proximal to distal. It may have affected the assessment of the actual degree of the supraspinatus muscle disease. In the current literature that we know of there is not a validated semiquantitative histopathologic classification of the supraspinatus muscle atrophy or fatty infiltration. We are aware that the classification system adopted in the present study may have some limitations. Moreover, despite their statistically significance, the two correlations of the present study are weak: further studies with larger sample size are needed in order to confirm these results. On the MRI, the tangent sign was used to judge supraspinatus muscle atrophy [27]: we are aware of the high risk of false positives because of the retraction from larger rotator cuff tears when using a relatively lateral sagittal view.

Referring to the radiological evaluation, given the fact that radiologists and surgeons do not agree well when analyzing images [60], a potential limitation of the present study is the lack of a Board Certified Radiologist among authors. A comparison between Orthopaedists and Radiologists in the assessment of MRI findings would have improved the study quality.

Finally, we are not aware of the level of rotator cuff tendon and muscle degeneration in the general Italian adult population. We are not aware of any study detailing the histological appearance of rotator cuff tendon degeneration in this population.

Conclusions

Microscopic tendon alterations occur together with muscle alterations in patients with RC tendinopathy. Muscle atrophy is a direct consequence of tendon detachment and an early diagnosis is mandatory in order to prevent irreversible changes. Microscopic alterations are intrinsically linked to clinical evidence of disease: findings of the present study suggest a major role of the muscle atrophy, rather than tendon degeneration and fatty infiltration, in impairing the supraspinatus clinical function. Shoulder pain seems to be independent from morphological tendon and muscle alterations and further studies are required in order to investigate its triggers. Radiological evaluation of the supraspinatus muscle may be not so accurate in determining the actual stage of the disease, increasing the risk of late diagnoses.

Availability of data and materials

The dataset supporting the conclusions of this article is included within the article.

Abbreviations

RC:

Rotator cuff

ROM:

Range of motion

MRI:

Magnetic resonance imaging

PBS:

Phosphate buffered saline

TBS:

Tris-Buffered saline

DAB:

Diaminobenzidine

VAS:

Visual analogue scale

SD:

Standard deviation

GAG:

Glycosaminoglycans

References

  1. Tashjian RZ. Epidemiology, natural history, and indications for treatment of rotator cuff tears. Clin Sports Med. 2012;31(4):589–604.

    Article  PubMed  Google Scholar 

  2. Longo UG, Salvatore G, Rizzello G, Berton A, Ciuffreda M, Candela V, et al. The burden of rotator cuff surgery in Italy: a nationwide registry study. Arch Orthop Trauma Surg. 2017;137(2):217–24.

    Article  PubMed  Google Scholar 

  3. Longo UG, Franceschi F, Ruzzini L, Rabitti C, Morini S, Maffulli N, et al. Histopathology of the supraspinatus tendon in rotator cuff tears. Am J Sports Med. 2008;36(3):533–8.

    Article  PubMed  Google Scholar 

  4. Hsu J, Keener JD. Natural History of Rotator Cuff Disease and Implications on Management. Oper Tech Orthop. 2015;25(1):2–9.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Carbone S, Gumina S, Arceri V, Campagna V, Fagnani C, Postacchini F. The impact of preoperative smoking habit on rotator cuff tear: cigarette smoking influences rotator cuff tear sizes. J Shoulder Elbow Surg. 2012;21(1):56–60.

    Article  PubMed  Google Scholar 

  6. Dunn WR, Kuhn JE, Sanders R, An Q, Baumgarten KM, Bishop JY, et al. Symptoms of pain do not correlate with rotator cuff tear severity: a cross-sectional study of 393 patients with a symptomatic atraumatic full-thickness rotator cuff tear. J Bone Joint Surg Am. 2014;96(10):793–800.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Longo UG, Mazzola A, Carotti S, Francesconi M, Catapano S, Magrì F, et al. The role of estrogen and progesterone receptors in the rotator cuff disease: a retrospective cohort study. BMC Musculoskelet Disord. 2021;22(1):891.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gibbons MC, Singh A, Anakwenze O, Cheng T, Pomerantz M, Schenk S, et al. Histological evidence of muscle degeneration in advanced human rotator cuff disease. J Bone Joint Surg Am. 2017;99(3):190–9.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Steinbacher P, Tauber M, Kogler S, Stoiber W, Resch H, Sanger AM. Effects of rotator cuff ruptures on the cellular and intracellular composition of the human supraspinatus muscle. Tissue Cell. 2010;42(1):37–41.

    Article  CAS  PubMed  Google Scholar 

  10. Chard MD, Cawston TE, Riley GP, Gresham GA, Hazleman BL. Rotator cuff degeneration and lateral epicondylitis: a comparative histological study. Ann Rheum Dis. 1994;53(1):30–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Riley GP, Harrall RL, Constant CR, Chard MD, Cawston TE, Hazleman BL. Tendon degeneration and chronic shoulder pain: changes in the collagen composition of the human rotator cuff tendons in rotator cuff tendinitis. Ann Rheum Dis. 1994;53(6):359–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hashimoto T, Nobuhara K, Hamada T. Pathologic evidence of degeneration as a primary cause of rotator cuff tear. Clin Orthop Relat Res. 2003;415:111–20.

    Article  Google Scholar 

  13. Kang JR, Gupta R. Mechanisms of fatty degeneration in massive rotator cuff tears. J Shoulder Elbow Surg. 2012;21(2):175–80.

    Article  PubMed  Google Scholar 

  14. Deniz G, Kose O, Tugay A, Guler F, Turan A. Fatty degeneration and atrophy of the rotator cuff muscles after arthroscopic repair: does it improve, halt or deteriorate? Arch Orthop Trauma Surg. 2014;134(7):985–90.

    Article  PubMed  Google Scholar 

  15. Valencia AP, Lai JK, Iyer SR, Mistretta KL, Spangenburg EE, Davis DL, et al. Fatty infiltration is a prognostic marker of muscle function after rotator cuff tear. Am J Sports Med 2018. https://doi.org/10.1177/0363546518769267.

  16. Bonaldo P, Sandri M. Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech. 2013;6(1):25–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Millstein ES, Snyder SJ. Arthroscopic management of partial, full-thickness, and complex rotator cuff tears: indications, techniques, and complications. Arthroscopy. 2003;19(Suppl 1):189–99.

    Article  PubMed  Google Scholar 

  18. Spencer EE, Dunn WR, Wright RW, Wolf BR, Spindler KP, McCarty E, et al. Interobserver agreement in the classification of rotator cuff tears using magnetic resonance imaging. Am J Sports Med. 2008;36(1):99–103.

    Article  PubMed  Google Scholar 

  19. Ellman H. Diagnosis and treatment of incomplete rotator cuff tears. Clin Orthop Relat Res. 1990;254:64–74.

    Article  Google Scholar 

  20. Longo UG, Berton A, Ahrens PM, Maffulli N, Denaro V. Clinical tests for the diagnosis of rotator cuff disease. Sports Med Arthrosc Rev. 2011;19(3):266–78.

    Article  PubMed  Google Scholar 

  21. Sternberger LA, Sternberger NH. The unlabeled antibody method: comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification. J Histochem Cytochem. 1986;34(5):599–605.

    Article  CAS  PubMed  Google Scholar 

  22. Cook JL, Feller JA, Bonar SF, Khan KM. Abnormal tenocyte morphology is more prevalent than collagen disruption in asymptomatic athletes’ patellar tendons. J Orthop Res. 2004;22(2):334–8.

    Article  CAS  PubMed  Google Scholar 

  23. Aström M, Rausing A. Chronic Achilles tendinopathy. A survey of surgical and histopathologic findings. Clin Orthop Relat Res 1995;(316):151–64.

  24. Longo UG, Saris D, Poolman RW, Berton A, Denaro V. Instruments to assess patients with rotator cuff pathology: a systematic review of measurement properties. Knee Surg Sports Traumatol Arthrosc. 2012;20(10):1961–70.

    Article  PubMed  Google Scholar 

  25. Longo UG, Vasta S, Maffulli N, Denaro V. Scoring systems for the functional assessment of patients with rotator cuff pathology. Sports Med Arthrosc Rev. 2011;19(3):310–20.

    Article  PubMed  Google Scholar 

  26. Hawker GA, Mian S, Kendzerska T, French M. Measures of adult pain: Visual Analog Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS), and Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP). Arthritis Care Res (Hoboken). 2011;63(Suppl 11):S240–52.

    Article  PubMed  Google Scholar 

  27. Zanetti M, Gerber C, Hodler J. Quantitative assessment of the muscles of the rotator cuff with magnetic resonance imaging. Invest Radiol. 1998;33(3):163–70.

    Article  CAS  PubMed  Google Scholar 

  28. Thomazeau H, Rolland Y, Lucas C, Duval JM, Langlais F. Atrophy of the supraspinatus belly. Assessment by MRI in 55 patients with rotator cuff pathology. Acta Orthop Scand. 1996;67(3):264–8.

    Article  CAS  PubMed  Google Scholar 

  29. Fuchs B, Weishaupt D, Zanetti M, Hodler J, Gerber C. Fatty degeneration of the muscles of the rotator cuff: assessment by computed tomography versus magnetic resonance imaging. J Shoulder Elbow Surg. 1999;8(6):599–605.

    Article  CAS  PubMed  Google Scholar 

  30. Goutallier D, Postel JM, Bernageau J, Lavau L, Voisin MC. Fatty muscle degeneration in cuff ruptures. Pre- and postoperative evaluation by CT scan. Clin Orthop Relat Res. 1994;304:78–83.

    Article  Google Scholar 

  31. Longo UG, Berton A, Khan WS, Maffulli N, Denaro V. Histopathology of rotator cuff tears. Sports Med Arthrosc Rev. 2011;19(3):227–36.

    Article  PubMed  Google Scholar 

  32. O’Brien M. Structure and metabolism of tendons. Scand J Med Sci Sports. 1997;7(2):55–61.

    Article  CAS  PubMed  Google Scholar 

  33. Józsa L, Bálint JB, Réffy A, Demel Z. Histochemical and ultrastructural study of adult human tendon. Acta Histochem. 1979;65(2):250–7.

    Article  PubMed  Google Scholar 

  34. Khan KM, Cook JL, Bonar F, Harcourt P, Astrom M. Histopathology of common tendinopathies. Update and implications for clinical management. Sports Med. 1999;27(6):393–408.

    Article  CAS  PubMed  Google Scholar 

  35. Fukuda H, Hamada K, Yamanaka K. Pathology and pathogenesis of bursal-side rotator cuff tears viewed from en bloc histologic sections. Clin Orthop Relat Res. 1990;254:75–80.

    Article  Google Scholar 

  36. Nixon JE, DiStefano V. Ruptures of the rotator cuff. Orthop Clin North Am. 1975;6(2):423–47.

    Article  CAS  PubMed  Google Scholar 

  37. Movin T, Kristoffersen-Wiberg M, Rolf C, Aspelin P. MR imaging in chronic Achilles tendon disorder. Acta Radiol. 1998;39(2):126–32.

    Article  CAS  PubMed  Google Scholar 

  38. Maffulli N, Regine R, Angelillo M, Capasso G, Filice S. Ultrasound diagnosis of Achilles tendon pathology in runners. Br J Sports Med. 1987;21(4):158–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Movin T, Kristoffersen-Wiberg M, Shalabi A, Gad A, Aspelin P, Rolf C. Intratendinous alterations as imaged by ultrasound and contrast medium-enhanced magnetic resonance in chronic achillodynia. Foot Ankle Int. 1998;19(5):311–7.

    Article  CAS  PubMed  Google Scholar 

  40. Chansky HA, Iannotti JP. The vascularity of the rotator cuff. Clin Sports Med. 1991;10(4):807–22.

    Article  CAS  PubMed  Google Scholar 

  41. Lohr JF, Uhthoff HK. The microvascular pattern of the supraspinatus tendon. Clin Orthop Relat Res. 1990;254:35–8.

    Article  Google Scholar 

  42. Biberthaler P, Wiedemann E, Nerlich A, Kettler M, Mussack T, Deckelmann S, et al. Microcirculation associated with degenerative rotator cuff lesions. In vivo assessment with orthogonal polarization spectral imaging during arthroscopy of the shoulder. J Bone Joint Surg Am. 2003;85(3):475–80.

    Article  PubMed  Google Scholar 

  43. Rathbun JB, Macnab I. The microvascular pattern of the rotator cuff. J Bone Joint Surg Br. 1970;52(3):540–53.

    Article  CAS  PubMed  Google Scholar 

  44. Karthikeyan S, Griffin DR, Parsons N, Lawrence TM, Modi CS, Drew SJ, et al. Microvascular blood flow in normal and pathologic rotator cuffs. J Shoulder Elbow Surg. 2015;24(12):1954–60.

    Article  PubMed  Google Scholar 

  45. Gigliotti D, Xu MC, Davidson MJ, Macdonald PB, Leiter JRS, Anderson JE. Fibrosis, low vascularity, and fewer slow fibers after rotator-cuff injury. Muscle Nerve. 2017;55(5):715–26.

    Article  CAS  PubMed  Google Scholar 

  46. Melis B, Wall B, Walch G. Natural history of infraspinatus fatty infiltration in rotator cuff tears. J Shoulder Elbow Surg. 2010;19(5):757–63.

    Article  PubMed  Google Scholar 

  47. Gladstone JN, Bishop JY, Lo IK, Flatow EL. Fatty infiltration and atrophy of the rotator cuff do not improve after rotator cuff repair and correlate with poor functional outcome. Am J Sports Med. 2007;35(5):719–28.

    Article  PubMed  Google Scholar 

  48. Gerber C, Schneeberger AG, Hoppeler H, Meyer DC. Correlation of atrophy and fatty infiltration on strength and integrity of rotator cuff repairs: a study in thirteen patients. J Shoulder Elbow Surg. 2007;16(6):691–6.

    Article  PubMed  Google Scholar 

  49. Cross SS. Kappa statistics as indicators of quality assurance in histopathology and cytopathology. J Clin Pathol. 1996;49(7):597–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Gibbons MC, Singh A, Engler AJ, Ward SR. The role of mechanobiology in progression of rotator cuff muscle atrophy and degeneration. J Orthop Res. 2018;36(2):546–56.

    PubMed  Google Scholar 

  51. Harris JD, Pedroza A, Jones GL, Group MMOONS. Predictors of pain and function in patients with symptomatic, atraumatic full-thickness rotator cuff tears: a time-zero analysis of a prospective patient cohort enrolled in a structured physical therapy program. Am J Sports Med. 2012;40(2):359–66.

    Article  PubMed  Google Scholar 

  52. Krief OP, Huguet D. Shoulder pain and disability: comparison with MR findings. AJR Am J Roentgenol. 2006;186(5):1234–9.

    Article  PubMed  Google Scholar 

  53. Wylie JD, Suter T, Potter MQ, Granger EK, Tashjian RZ. Mental health has a stronger association with patient-reported shoulder pain and function than tear size in patients with full-thickness rotator cuff tears. J Bone Joint Surg Am. 2016;98(4):251–6.

    Article  PubMed  Google Scholar 

  54. Escamilla RF, Yamashiro K, Paulos L, Andrews JR. Shoulder muscle activity and function in common shoulder rehabilitation exercises. Sports Med. 2009;39(8):663–85.

    Article  PubMed  Google Scholar 

  55. Colachis SC, Strohm BR. Effect of suprascauular and axillary nerve blocks on muscle force in upper extremity. Arch Phys Med Rehabil. 1971;52(1):22–9.

    PubMed  Google Scholar 

  56. De Duca CJ, Forrest WJ. Force analysis of individual muscles acting simultaneously on the shoulder joint during isometric abduction. J Biomech. 1973;6(4):385–93.

    Article  PubMed  Google Scholar 

  57. Otis JC, Jiang CC, Wickiewicz TL, Peterson MG, Warren RF, Santner TJ. Changes in the moment arms of the rotator cuff and deltoid muscles with abduction and rotation. J Bone Joint Surg Am. 1994;76(5):667–76.

    Article  CAS  PubMed  Google Scholar 

  58. Kuzel BR, Grindel S, Papandrea R, Ziegler D. Fatty infiltration and rotator cuff atrophy. J Am Acad Orthop Surg. 2013;21(10):613–23.

    Article  PubMed  Google Scholar 

  59. Fu MC, O’Donnell EA, Taylor SA, Aladesuru OM, Rauck RC, Dines JS, et al. Delay to arthroscopic rotator cuff repair is associated with increased risk of revision rotator cuff surgery. Orthopedics. 2020;43(6):340–4.

    Article  PubMed  Google Scholar 

  60. Zingg T, Uldry E, Omoumi P, Clerc D, Monier A, Pache B, et al. Interobserver reliability of the Tile classification system for pelvic fractures among radiologists and surgeons. Eur Radiol. 2021;31(3):1517–25.

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Research Unit of Orthopaedic and Trauma Surgery, Fondazione Policlinico Universitario Campus Bio-Medico, Via Alvaro del Portillo, 200 - 00128, Roma, Italy

    Umile Giuseppe Longo, Alessandro Mazzola, Francesco Magrì, Simone Catapano, Sergio De Salvatore & Vincenzo Denaro

  2. Research Unit of Orthopaedic and Trauma Surgery, Department of Medicine and Surgery, Università Campus Bio-Medico Di Roma, Via Alvaro del Portillo, 21 - 00128, Roma, Italy

    Umile Giuseppe Longo, Alessandro Mazzola, Francesco Magrì, Simone Catapano, Sergio De Salvatore & Vincenzo Denaro

  3. Unit of Microscopic and Ultrastructural Anatomy, University Campus Bio-Medico, Rome, Italy

    Simone Carotti

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Contributions

UGL, AM: manuscript preparation, study design, database interpretation and manuscript revision. UGL, AM: manuscript preparation, database interpretation and statistical analysis. AM, FM, SDS: manuscript preparation, figures and tables preparation, study design. UGL, AM, SC, SDS: Manuscript preparation and database interpretation. UGL, SDS, VD: Study design, manuscript revision. The author(s) read and approved the final manuscript.

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Correspondence to Umile Giuseppe Longo.

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The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Campus Bio-Medico University of Rome (COSMO study, Protocol number: 78/18 OSS ComEt CBM, 16/10/18). Informed consent was obtained from all participants. 

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UGL is a member of the Editorial Board of BMC Musculoskeletal Disorders. The remaining authors declare that they have no conflict of interest.

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Longo, U., Mazzola, A., Magrì, F. et al. Histological, radiological and clinical analysis of the supraspinatus tendon and muscle in rotator cuff tears. BMC Musculoskelet Disord 24, 127 (2023). https://doi.org/10.1186/s12891-023-06237-9

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BMC Musculoskeletal Disorders

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