Native Enthesis Preservation Versus Removal in Rotator Cuff Repair in a Rabbit Model.

PURPOSE: The purpose of this study was to explore whether preservation of native enthesis during rotator cuff repair (RCR) results in a histologically and biomechanically better rotator cuff-greater tuberosity (RC-GT) connection in an animal model. METHODS: Thirty-six New Zealand white rabbits were used in this study. The supraspinatus tendons were cut from the footprint to create a rotator cuff tear (RCT) on both shoulders, followed by immediate repair, with a 1 mm thick tendon stump remaining on the footprint. On one side, RCR was performed by attaching the medial rotator cuff to the GT over the footprint stump layer (enthesis preservation [EP] approach). On the other side, the footprint stump and enthesis were fully removed and the footprint was decorticated. The repair was performed by attaching the medial rotator cuff onto the spongy bone surface in the footprint area (enthesis removal [ER] approach). Twelve rabbits were sacrificed at 4, 8, and 12 weeks, of which 6 were used for histological analysis and 6 for biomechanical evaluation. RESULTS: Histological examination revealed that in the EP group, the native enthesis healed well with the reattached cuff tendon, and continuous collagen crossed the tendon-tendon interface area at 12 weeks. However, in the ER group, there was only fibrous scar tissue formation at the tendon-bone interface. Biomechanical testing revealed a significantly higher load to failure (P = .001) of the RC-GT structure in the EP group than that in the ER group at 12 weeks. CONCLUSIONS: For acute RCT with remnant enthesis, EP-RCR incorporates the native enthesis functionally in the restored RC-GT connection and enhances the RC-GT connection when compared with ER-RCR at 12 weeks in a rabbit model. CLINICAL RELEVANCE: This finding justifies a human trial because in RCR with a remnant footprint, the EP technique may be adopted for a better RC-GT connection.

Novel engineered tendon-fibrocartilage-bone composite with cyclic tension for rotator cuff repair.

Surgical repair of rotator cuff tears presents a significant clinical challenge with high failure rates and inferior functional outcomes. Graft augmentation improves repair outcomes; however, currently available grafting materials have limitations. Although cell-seeded decellularized tendon slices may facilitate cell infiltration, promote tendon incorporation, and preserve original mechanical strength, the unique fibrocartilage zone is yet to be successfully reestablished. In this study, we investigated the biological and mechanical properties of an engineered tendon-fibrocartilage-bone composite (TFBC) with cyclic tension (3% strain; 0.2 Hz). Decellularized TFBCs seeded with bone marrow-derived mesenchymal stem cell (BMSCs) sheets and subjected to mechanical stimulation for up to 7 days were characterised by histology, immunohistochemistry, scanning electron microscopy, mechanical testing, and transcriptional regulation. The decellularized TFBC maintained native enthesis structure and properties. Mechanically stimulated TFBC-BMSC constructs displayed increased cell migration after 7 days of culture compared with static groups. The seeded cell sheet not only integrated well with tendon scaffold but also distributed homogeneously and aligned to the direction of stretch under dynamic culture. Developmental genes were regulated including scleraxis, which was significantly upregulated with mechanical stimulation. The Young's modulus of the cell-seeded constructs was significantly higher compared with the noncell-seeded controls. In conclusion, the results of this study reveal that the TFBC-BMSC composite provides an ideal multilayer construct for cell seeding and growth, with mechanical preconditioning further enhances cell penetration and differentiation. The BMSC cell sheet revitalised TFBC in conjunction with mechanical stimulation could serve as a novel and primed biological patch to improve rotator cuff repair.

More histologic and ultrastructural degenerative signs in the subscapularis tendon and the joint capsule in male patients with shoulder impingement.

PURPOSE: The purpose of the present study was to analyze biopsy samples from the subscapularis tendon and from the joint capsule from male patients with shoulder impingement syndrome (SAIS) and compare them with samples from male patients with post-traumatic recurrent shoulder instability. The hypothesis of the study was that patients with SAIS would have more histologic and ultrastructural degenerative changes in their subscapularis tendon and joint capsule than patients with post-traumatic recurrent shoulder instability. METHODS: Male patients scheduled for surgery, with either subacromial decompression or Bankart reconstruction, were included. Four biopsies from each patient were obtained from the capsule and four from the subscapularis tendon during arthroscopic surgery. The histologic characteristics and the presence of glycosaminoglycans were assessed using the light microscope, and the ultrastructure was assessed using a transmission electron microscope. RESULTS: Eight patients, median age 53 (45-74) years (p < 0.0001), were included in the impingement group, and 12 patients, median age 27 (22-48) years, were included in the instability group. The histologic assessment revealed significantly higher cellularity and total degeneration score in the capsule (p = 0.016 and p = 0.014 respectively) in patients with subacromial impingement compared with the instability patients. The corresponding finding was not made for the subscapularis tendon. The ultrastructural evaluation revealed that the instability patients had more fibrils with a large diameter (indicating less degeneration) in both the subscapularis tendon and the capsule compared with the impingement patients (p < 0.0001). CONCLUSION: Male patients with subacromial impingement have more histologic and ultrastructural degenerative changes in their shoulder compared with patients with post-traumatic recurrent shoulder instability. CLINICAL RELEVANCE: It appears that in patients with subacromial impingement, the whole shoulder joint is affected and not only the subacromial space. It is the opinion of the authors that intra-articular therapeutic injections could be tried more often in these patients. LEVEL OF EVIDENCE: III.

The tendon-to-bone transition of the rotator cuff: a preliminary Raman spectroscopic study documenting the gradual mineralization across the insertion in rat tissue samples.

We applied Raman spectroscopy to monitor the distribution of mineral and the degree of mineralization across the tendon-bone insertion site in the shoulders of five rats. We acquired Raman spectra from 100 to 4,000 Deltacm(-1) on individual 1 microm points across the 120 microm wide transition zone of each tissue sample and identified all the peaks detected in pure tendon and in pure bone, as well as in the transition zone. The intensity of the 960 Deltacm(-1) P-O stretch for apatite (normalized to either the 2,940 Deltacm(-1) C-H stretch or the 1,003 Deltacm(-1) C-C stretch for collagen) was used as an indicator of the abundance of mineral. We relate the observed histological morphology in the tissue thin section with the observed Raman peaks for both the organic component (mostly collagen) and the inorganic component (a carbonated form of the mineral apatite) and discuss spectroscopic issues related to peak deconvolution and quantification of overlapping Raman peaks. We show that the mineral-to-collagen ratio at the insertion site increases linearly (R(2) = 0.8 for five samples) over the distance of 120 microm from tendon to bone, rather than abruptly, as previously inferred from histological observations. In addition, narrowing of the 960 Deltacm(-1) band across the traverse indicates that the crystalline ordering within the apatite increases concomitantly with the degree of mineralization. This finding of mineral gradation has important clinical implications and may explain why the uninjured tendon-to-bone connection of the rotator cuff can sustain very high loads without failure. Our finding is also consistent with recent mechanical models and calculations developed to better understand the materials properties of this unusually strong interface.

Supraspinatus tendon composition remains altered long after tendon detachment.

Most rotator cuff surgery is performed on chronic tears, but changes in the composition of chronically torn tendons remain poorly understood. In this study we surgically created supraspinatus tears in the rat and analyzed the composition of the tendon over time using immunohistochemistry. We found that collagen types I and XII were greatly increased initially after injury and then decreased with time. Collagen type III was detected and persisted in the scar for months. Decorin and biglycan were increased initially and then decreased, although decorin remained elevated from normal for months after injury. Aggrecan and collagen type II were detected in small amounts after detachment, which was associated with the expression of sulfated glycosaminoglycans. These alterations were similar to those seen in human studies. As the quality of the tendon is an important factor in repair, these findings may partially explain why chronic tears heal differently than acute tears.

The anatomy of the subscapularis tendon insertion as applied to rotator cuff repair.

The tendons constituting the rotator cuff (RC) are often torn, and several techniques for their repair have been established. The existence of an extension of the supraspinatus tendon into that of the subscapularis has often been overlooked. The purpose of this study was to study this extension in human dissections. The possible posterolateral extension of the subscapularis tendon and the interdigitating of this extension with the supraspinatus tendon were addressed. A horizontal band of tissue 1 cm below the superior ridge of the greater tuberosity of the humerus was harvested from 43 cadavers and thus included tissue constituting the greater tuberosity, bicipital groove, and lesser tuberosity. The sample extended 2 cm medially along the lesser tuberosity and 1 cm laterally along the greater tuberosity. Macroscopic findings suggest a continuous band of tissue extending across the bicipital groove. High collagen content was microscopically confirmed at different sections, and the collagen was densely distributed along the horizontal axis of the tissue samples.

Fibronectin, MMP-1 and histologic changes in rotator cuff disease.

This study was designed to investigate human surgical specimens from patients with impingement (n = 16), ruptured supraspinatus tendons (n = 7), frozen shoulder (n = 2) and controls (n = 9) with respect to histological changes and the presence of fibronectin and Matrix metalloprotease-1 (MMP-1). The biopsy of the middle part of the supraspinatus tendons was analyzed microscopically after staining with hematoxyline eosin, Van Giesons hematoxyline and Phospho Tungstic Acid Hematoxyline for visualization of fibrin. Immunofluorescent stainings for fibronectin and MMP-1 were performed. Histology and immunofluorescence were assessed blindly. Necrotic tendinous tissue and fibrin were found only in some specimens from ruptures. The staining for fibronectin was significantly increased among patients with a rupture. MMP-1 was, however, only infrequently found in specimens from patients with impingement and ruptures. Fibrosis and thinning of fascicles seemed to be a more non-specific finding, appearing in control, impingement and rupture specimens. In conclusion, necrotic tendinous tissue, fibrin and fibronectin appear to be signs of tendon degeneration, whereas fibrosis and thinning of fascicles were found also in controls.

Carrageenan-induced subacromial bursitis caused changes in the rat's rotator cuff.

This study was designed to investigate the histologic expression of the rat's supra- and infraspinatus tendons in carrageenan-induced subacromial bursitis. Thirty-two rats received subacromial injections with carrageenan (n = 28) or saline (n = 4). The tendons were analysed microscopically after staining with hematoxyline eosin, Van Giesons hematoxyline and immunofluorescent staining of fibronectin and fibrinogen. In the controls (saline x 10) and group A (carrageenan x 5) there were no changes in the tendons. In group B (carrageenan x 10) 3/8 rats showed macrophages between the collagen fibres and an increased staining of fibronectin. In group C (double dosis carrageenan) all rats had signs of fibrocartilaginous metaplasia in the supraspinatus tendon. In eight of these specimens even bony metaplasia was seen. The infraspinatus tendon showed fibrosis but no fibrocartilaginous metaplasia. The results showed that iatrogenic bursitis after carrageenan subacromial injections was associated with marked changes of the supraspinatus tendon.

Significance of granulation tissue in torn supraspinatus insertions: an immunohistochemical study with antibodies against interleukin-1 beta, cathepsin D, and matrix metalloprotease-1.

The pathophysiology of rotator cuff tears can be elucidated by examining the tendinous insertion of the supraspinatus muscle. As seen by light microscopy, the granulation tissue around the insertion of a torn supraspinatus tendon appears to induce osteochondral destruction by means of multinucleated giant cells and chemical mediators. The purpose of this study was to examine the contribution of certain chemical mediators to osteochondral destruction using immunohistochemical analysis of interleukin-beta, cathepsin D, and matrix metalloprotease-1. Sixteen supraspinatus insertions with portions of the greater tuberosity, including eight complete-thickness tears and eight incomplete-thickness tears, were obtained during surgery. Six fresh cadaveric supraspinatus tendons without grossly evident tears served as normal controls. Strong immunoreactivity was found in all 16 torn supraspinatus insertions but not in the six insertions of apparently intact tendons. Macrophages and multinucleated giant cells, which showed immunoreactivity for all three chemical mediators, were often found at the interface between the osteochondral margin of the enthesis and the granulation tissue, suggesting that they may be involved in osteochondral destruction. We therefore concluded that, in addition to repetitive subacromial impingement, this granulation tissue may contribute to the development of rotator cuff tears by weakening the insertion.

The collagen types in the attachment zone of rotator cuff tendons in the elderly: an immunohistochemical study.

OBJECTIVE: The attachment zone of the rotator cuff tendons in the elderly was studied immunohistochemically in order to determine how degenerative changes affected the pattern of collagen fiber distribution. METHODS: Twenty-seven cuffs with their bony insertion were obtained from 22 postmortem cases of both sexes ranging in age from 52 to 90 years and without a history of shoulder ailments. In addition, 3 cuff specimens from cadavers in the 3rd and 4th decades were examined for comparison. Sections of formalin fixed tissues were stained by peroxidase antiperoxidase (PAP) technique using monoclonal antibodies against types I, II and III collagen. RESULTS: Degenerative changes affecting the fibrocartilage primarily were characterized by calcification, fibrovascular proliferation and microtears. In addition, they were found in all the cuff tendons of elderly individuals but not in those from younger subjects. Immunohistochemically, the attachment zone in areas without degenerative lesions showed collagen type I labelling strongly in bone but only moderately in the fibrocartilage. The predominant labelling in the fibrocartilage was for collagen type II, and collagen type III labelled principally in perichondrocytic areas. The tide-mark showed inconsistent labelling for any of the collagen types. In the presence of degenerative lesions, the disposition of fiber types was interrupted by calcification and microtears. Collagen type II composition of the fibrocartilage was markedly altered by the presence of fibrovascular tissue which labelled only for collagen type III. CONCLUSION: We conclude that severe degenerative changes in the cuff tendons of elderly individuals, alter the collagen characteristic of the rotator cuff and that the changes could be associated with impairment of biomechanical properties of the attachment zone, and may give rise to the clinical syndrome of enthesopathy.

Glycosaminoglycans of human rotator cuff tendons: changes with age and in chronic rotator cuff tendinitis.

OBJECTIVES: To analyse the glycosaminoglycans of the adult human rotator cuff tendon matrix, to characterise changes in the glycosaminoglycan composition with age and in chronic rotator cuff tendinitis. METHODS: Rotator cuff (supraspinatus) tendons (n = 84) and common biceps tendons (n = 26) were obtained from cadavers with no history of tendon pathology (age range 11-95 years). Biopsies of rotator cuff tendons (supraspinatus and subscapularis tendons, n = 53) were obtained during open shoulder surgery to repair shoulder lesions (age range 38-80 years). Glycosaminoglycans were extracted by papain digestion and analysed by cellulose acetate electrophoresis, the carbazole assay for uronic acid and the dimethylmethylene blue dye-binding assay for sulphated glycosaminoglycans. Some digests were analysed for keratan sulphate by 5D4 monoclonal antibody ELISA. Soluble proteoglycans were extracted in 4M guanidine hydrochloride and analysed by 4-15% SDS PAGE. RESULTS: The mean (SD) sulphated glycosaminoglycan (GAG) content of the normal cadaver supraspinatus tendon was 12.3 (4.3) micrograms/mg dry weight, between three and ten times greater than in the common biceps tendon [1.2 (0.6) micrograms/mg dry weight]. The major GAG was chondroitin sulphate [6.9 (2.6) micrograms/mg dry weight], with a smaller proportion of dermatan sulphate [2.5 (1.2) micrograms/mg dry weight]. In contrast, the common biceps tendon contained predominantly dermatan sulphate [0.8 (0.2) microgram/mg dry weight] with less chondroitin sulphate [0.2 (0.2) microgram/mg dry weight]. There was no difference in the concentration of hyaluronan in these tendons [9.3 (2.8) micrograms/mg dry weight and 10.8 (4.3) micrograms/mg dry weight respectively] and there was no significant change of hyaluronan with age. Keratan sulphate was a small but significant component of the supraspinatus tendon [0.43 (0.33) microgram/mg dry weight, n = 25], whereas there was little or none in the common biceps tendon [0.04 (0.05) microgram/mg dry weight, n = 8] and there was no significant change across the age range. In the supraspinatus tendon, there was a significant decrease in total glycosaminoglycan, chondroitin sulphate and dermatan sulphate with age (p < 0.001), whether expressed relative to the tendon dry weight or total collagen content, and no change in the relative proportion of the different GAG types. There was, however, a large degree of variation within the samples. Supraspinatus tendons from patients with chronic tendinitis had a significantly increased concentration of hyaluronan [30.4 (10.1) micrograms/mg dry weight, p < 0.001], chondroitin sulphate [8.4 (1.8) micrograms/mg dry weight, p < 0.05] and dermatan sulphate [3.8 (1.1) micrograms/mg dry weight, p < 0.001] compared with normal cadaver supraspinatus tendons, although the keratan sulphate content was not significantly different [0.18 (0.05) microgram/mg dry weight]. CONCLUSIONS: The normal supraspinatus tendon has the proteoglycan/glycosaminoglycan of tendon fibrocartilage, which it is suggested is an adaptation to mechanical forces (tension, compression and shear) which act on the rotator cuff tendons in the shoulder, although other factors such as reduced vascularity, low oxygen tension and the influence of local growth factors may also be important. This functional adaptation may have important consequences for the structural strength of the supraspinatus tendon and to influence the ability of the tendon to repair after injury. The glycosaminoglycan composition of tendon specimens from patients with chronic tendinitis is consistent with acute inflammation and new matrix proteoglycan synthesis, even in relatively old tendon specimens and after at least one injection of corticosteroid.