Rotator Cuff Tear Degeneration and Cell Apoptosis in Smokers vs Nonsmokers

Abstract

Purpose

The purpose of this study was to assess the effect of smoking on supraspinatus tendon degeneration, including cellular alterations, proliferation, and apoptosis of tendon cells.

Methods

Supraspinatus tendon samples of 10 smokers and 15 nonsmokers with full-thickness tears were compared, focusing on the severity of tendon histopathology including apoptosis (programmed cell death), cellularity, and proliferation. Immunohistochemistry was used to assess the density of apoptotic cells and proliferation. The extent of tendon degeneration was classified according to a revised version of the Bonar tendon histopathology score.

Results

The smokers were younger (P = .01). The symptom duration among smokers was longer (P < .05). The supraspinatus tendons from the smokers presented significantly more advanced degenerative changes (Bonar score, 13.5 [interquartile range, 1.4] v 9 [interquartile range, 3]; P < .001). The smokers’ tendons showed increased density of apoptotic cells (0.108 [SE, 0.038] v 0.0107 [SE, 0.007]; P = .024) accompanied by reduced tenocyte density (P = .019) and upregulation of proliferative activity (P < .0001).

Conclusions

Smoking is associated with worsened supraspinatus tendon histopathology and increased apoptosis.

Clinical Relevance

Pronounced degenerative changes, reduced tendon cellularity, and increased apoptosis may indicate reduced tendon healing capacity in smokers.

Introduction

Animal studies have shown negative effects of nicotine and cigarette smoking on the biomechanical properties of mesenchymal stem cells and tendon tissue,^{1 and 2} reduced fibroblast migration and survival,³ and impaired tendon healing.^{4 and 5} Human studies including a cadaveric study indicate smoking to be a risk factor for early development of rotator cuff tears and to be associated with reduced postoperative functional outcome.^{6, 7, 8, 9 and 10} However, the underlying mechanism of these effects, particularly the influence of smoking on tendon cells, is poorly understood.

Tendinosis is defined as structural tendon changes that are primarily of a degenerative nature and that are associated with histologic features consistent with a failed healing response.¹¹ Tendinosis tissue displays regions of both hypercellularity and hypocellularity, that is, a dysregulation of cell density, which could interfere with the assembly and maintenance of a healthy, load-bearing extracellular matrix. In human patellar tendon, regions that show increased matrix remodeling also show higher rates of tenocyte apoptosis and proliferation.¹² Apoptosis is an irreversible, physiological process of cell death that regulates cell density during development and adulthood and that is also associated with degenerative pathologies such as Alzheimer disease and osteoarthritis.^{13, 14, 15, 16 and 17}

The purpose of this study was to assess the effect of smoking on supraspinatus tendon degeneration, including cellular alterations, proliferation, and apoptosis of tendon cells. Our hypothesis was that smokers would exhibit more advanced degenerative changes, increased apoptosis, and reduced tendon cell proliferation compared with nonsmokers.

Methods

Patients

Twenty-five patients admitted for surgery for symptomatic supraspinatus tendon tears were included in this study. They were divided into 2 groups based on their smoking history. The group of smokers comprised 10 patients who fulfilled the inclusion criterion of smoking at least 10 cigarettes per day. The nonsmoking group comprised 15 patients not using any kind of nicotine supplement (e.g., nicotine gum or patch, oral snuff/moist snuff, and cigars) including cigarettes. The exclusion criteria for both groups were as follows: subscapularis tendon pathology, fatty infiltration of the muscle exceeding grade 1 according to Goutallier et al.,¹⁸ diabetes, and/or systemic inflammatory disorders.

Surgery was strictly arthroscopic and was performed under general anesthesia with the patient in the lateral decubitus position. Tear size was classified according to Post et al.¹⁹ by the operating surgeon. The samples were collected arthroscopically from the edge of the supraspinatus tear with a 3-mm biopsy punch. All patients underwent arthroscopic rotator cuff repair with a single-row technique.

The samples were fixed in fresh 10% buffered formalin for 16 to 24 hours at 4°C and then subsequently dehydrated and paraffin embedded for histology and immunohistochemistry analysis. The stained samples were scanned and evaluated with an Aperio ScanScope XT (Aperio Technologies, Vista, CA). They were evaluated for tendon degeneration, tenocyte density and proliferation, apoptosis, and p53 expression by examiners who were blinded to the sample identity.

The study was approved by the regional committee for research ethics (Helse Sør-Øst, 1.2007.728). Written informed consent was obtained.

Evaluation of Tendon Degeneration

Hematoxylin-eosin stains of 5-μm sections of tissue samples were prepared for the evaluation of morphology. Alcian blue stains were performed for identification of sulfated glycosaminoglycans. The extent of tendinosis was evaluated according to a revised version of the modified Bonar score.^{20 and 21} The modified Bonar score encompasses a semiquantitative scale that focuses on 4 diagnostic features of tendinosis: fibroblastic alterations (hypercellularity/hypocellularity), increased glycosaminoglycans, collagen disorganization or disarray, and hypervascularity or vascular remodeling. This revised score adds an evaluation of cell morphology, calcifications, and intratendinous adipocytes to the original score. According to this revised score, a completely normal tendon would score 0 and a maximally degenerated tendon would score 20. The evaluation was conducted by 2 observers in consensus, blinded to the sample identity. The validity and reliability of the modified Bonar score have been established previously.²² The intertester reliability of the revised version of the modified Bonar score is good (r2 = 0.71) according to Fearon et al.²¹

Evaluation of Proliferation

Cell density (cell number per square millimeter) was calculated after cell counts on hematoxylin-eosin stains and area measurement using the tracing tool in Aperio Imagescope software (Aperio Technologies).

Reagents and antibodies for Ki-67 immunohistochemistry were obtained from Ventana Medical Systems (Tucson, AZ). Staining was carried out with an automated immunohistochemistry unit (Discovery XT; Ventana Medical Systems) with a labeled streptavidin-biotin kit according to the manufacturer’s instructions, by use of a universal secondary antibody with 3,3′-diaminobenzidine as the substrate. Tissue sections were pretreated with EDTA buffer (CC1). The primary antibody was incubated at 37°C for 60 minutes and the secondary at 37°C for 30 minutes. Human formalin-fixed paraffin-embedded tonsil was used for optimization and positive control.

The proliferation index was calculated to estimate tendon proliferative activity. It was defined as the percentage of Ki-67–positive cells within all fields of a given biopsy specimen that showed positive labeling with the assay.

Detection of Apoptosis

Caspase 3

Apoptosis was assessed through the identification of tendon cells containing active (cleaved) caspase 3, which constitutes the final common executioner caspase for the apoptotic pathways.²³ Active caspase 3 was detected by a polyclonal antibody (Asp 175; Cell Signaling Technology, Beverly, CA). Tissue sections were deparaffinized in xylene and then washed sequentially in 100%, 95%, and 70% ethanol and phosphate-buffered saline solution. Slides were incubated in phosphate-buffered saline solution containing 0.1% NP-40 for 10 minutes, followed by 20-μg/mL proteinase K for 40 minutes. They were washed in distilled water and transferred to 60°C 50% formamide (vol/vol distilled water) for 20 minutes. Endogenous peroxidase activity was then blocked as described earlier, followed by further blocking in goat serum for 20 minutes and in avidin D and biotin solutions for 15 minutes each. The sections underwent permeabilization with methanol for 10 minutes. Endogenous peroxidases were quenched with 3% hydrogen peroxide. The sections were incubated with the primary antibody (1:50) overnight at 4°C and then visualized using an avidin-biotin complex kit with diaminobenzidine as the substrate (Vector Laboratories, Burlingame, CA) and Harris hematoxylin as the counterstain. Formaldehyde-fixed mammary rat tissue with or without the primary antibody was used as positive and negative controls.

The average number of caspase 3–positive cells per square millimeter was used to express the amount of apoptosis. The sample area (in square millimeters) was calculated using the tracing tool in Aperio Imagescope software.

p53

To further assess apoptosis, we examined the presence of p53 protein. A monoclonal antibody (M7001; Dako, Carpinteria, CA) was used in conjunction with automated immunohistochemistry (Ventana Medical Systems) as described earlier. Heat-mediated antigen retrieval in EDTA buffer (CC1) was performed. The primary antibody was incubated at 37°C for 60 minutes at a 1:100 dilution, followed by a secondary antibody at 37°C for 30 minutes. Human formalin-fixed breast cancer tissue was used for optimization and as a positive control. The percentage of p53-positive cells within all fields of a given biopsy specimen that showed positive labeling with the assay was calculated.

Statistical Analysis

For all the antibodies used, only nonvascular cells of fibroblastic appearance, embedded within the tendon extracellular matrix, were counted and analyzed. The Mann-Whitney U test was applied to compare the histologic and immunohistochemical data of smokers’ and nonsmokers’ tendons; P < .05 was considered significant. The t test for independent variables was used to compare age between the 2 groups, and the Fisher exact test was used to compare the distribution of small and medium tears between the groups. The Mann-Whitney U test was used to compare the chronicity of symptoms between smokers and nonsmokers. Data were analyzed by use of online statistical software available through VassarStats (Vassar College, Poughkeepsie, NY). Reliability testing and correlation of variables within patients were conducted with the Pearson correlation coefficient.

Results

Patients

Patient demographic data are shown in Table 1. The smokers were significantly younger (P = .01). They smoked a median of 15 cigarettes per day (range, 10 to 30). The symptom duration before surgery was significantly longer among smokers (Table 1). There was no statistically significant difference in the distribution of small- and medium-sized tears between smokers and nonsmokers (P = .18). In 1 smoker a postoperative infection developed that responded to 2 arthroscopic revisions and antibiotics. The implants were not removed. The tear healed with a residual defect smaller than the original tear.

Table 1.

Demographic Data of Patient Groups

Feature	Smokers	Nonsmokers
No. of cases	10	15
Mean age (SD) (yr)	52 (6.4)*	58 (5.8)
Sex (male/female)	8/2	10/5
Dominant shoulder affected	7	11
History of trauma	3	4
Median symptom duration (range) (mo)	19 (6–36)*	11 (1.5–72)
Tear size classification (small [<1 cm]/medium [1–3 cm])	4/6	2/13
Muscle changes on magnetic resonance imaging (atrophy/fatty infiltration)	4/0	8/1

^*Significant difference (P < .05).

Tendon degeneration

The histologic evaluation of tendon degeneration showed that the smokers had a greater degree of degeneration than the nonsmokers (Fig 1). Despite the fact that the smokers in this study tended to present with smaller tears, the Bonar score of the supraspinatus tendon was significantly increased.

Figure 1.

Tendon Proliferation

The supraspinatus tendons from the smokers showed a significant decrease in tendon cell density accompanied by an increased proliferation index (P < .001 for both indicators) (Fig 2 and Fig 3).

Figure 2.

Figure 3.

Apoptosis

The number of apoptotic cells per square millimeter was significantly increased among the smokers compared with the nonsmokers (P = .024) (Fig 4). The expression of protein p53 was also significantly stronger in the smokers, confirming a role for p53-dependent apoptosis in tendinopathy (median of 5 [interquartile range, 3.6] in smokers and median of 0 [interquartile range, 1.8] in nonsmokers; P < .0001).

Figure 4.

Discussion

To our knowledge, this is the first study to assess the effect of smoking on the histopathology of human tendons, including cellular alterations, proliferation, and apoptosis. We found smokers to be significantly younger but with a longer symptom duration. Their torn supraspinatus tendons showed more pronounced degeneration with associated reduced cellularity, increased proliferation, and increased apoptosis. Our findings are in line with previous clinical reports indicating that smokers are more prone to early-onset rotator cuff tendinopathy, reduced functional outcome after repair, and reduced healing ability after repair.^{6, 7, 8, 9 and 10} Smoking has been shown to decrease collagen synthesis and alter the extracellular matrix turnover in human skin fibroblasts in vivo, which also supports our findings.^{24 and 25} Previous publications of increased apoptosis in rotator cuff tendinopathy vary in their estimates on the extent of apoptosis.^{26, 27, 28 and 29} Our findings indicate that smoking needs to be controlled for in studies on tendinopathy; the inconsistency regarding information about smoking status in previous reports may contribute to the reported discrepancies in the extent of tenocyte apoptosis. Interestingly, tendon samples from smokers showed an increase in tenocyte proliferative activity accompanying the reduced cell density. This may illustrate an unsuccessful, compensatory attempt to restore tendon cell density and tendon mechanical properties. It may also indicate that the stimulation potential of tendon cells is preserved despite smoking.

Patients with tendinopathy and treating physicians need to appreciate the negative effects of smoking on tendon quality. Smoking cessation appears to be mandatory to obtain the best structural and functional results of rotator cuff repair.

Whether the negative effects of smoking are due to nicotine or other substances contained in cigarettes is not clear. Nicotine and carbon monoxide decrease microperfusion and tissue oxygenation, leading to tissue hypoxia,^{30, 31 and 32} and lower cellular oxygen levels are associated with increased tendon degeneration.^{27, 33 and 34} However, there are studies indicating that nicotine as such is not responsible for the negative effects on the musculoskeletal system.^{35, 36, 37, 38 and 39} Our findings reinforce the need for further research on the effects of smoked tobacco versus medicinal nicotine (used in nicotine replacement therapy) and smokeless tobacco products on the musculoskeletal system. This knowledge may guide nicotine-addicted patients to choose the safest nicotine product in terms of a harm-reduction strategy when cessation is impossible.

Limitations

Our findings are to be interpreted with caution because of the small number of patients. The difference in symptom duration before surgery between the 2 groups may also be a confounding factor. The dose-dependency rate cannot be estimated from this study. Our inclusion criterion of 10 cigarettes per day was chosen on the background of a previous publication, in which this dose was correlated to increased occurrence of shoulder pain.⁴⁰ Whether the identified effects are mitigated by nicotine or not may not be identified by this study model.

Conclusions

Smoking is associated with worsened supraspinatus tendon histopathology and increased apoptosis.