In Vivo Imaging of Fibroblast Activity Using a 68Ga-Labeled Fibroblast Activation Protein Alpha (FAP-α) Inhibitor: Study in a Mouse Rotator Cuff Repair Model.

BACKGROUND: Rotator cuff repair site failure is a well-established clinical concern. Tendon-to-bone healing is initiated by inflammatory mediators followed by matrix synthesis by fibroblasts. The kinetics of fibroblast accumulation and activity are currently poorly understood. METHODS: Ninety-six mice underwent supraspinatus tendon repair. Six were used for imaging using a novel 68Gallium (Ga)-labeled fibroblast activation protein alpha (FAP-α) inhibitor and positron emission tomography-computed tomography (PET/CT) at days 0 (before surgery), 3, 7, 14, and 28. Sixty-eight animals were divided into 4 groups to be evaluated at 3, 7, 14, or 28 days. Twenty-two native shoulders from mice without surgery were used as the control group (intact tendon). Six animals from each group were used for histological analysis; 6 from each group were used for evaluation of fibroblastic response-related gene expression; and 10 mice each from the intact, 14-day, and 28-day groups were used for biomechanical testing. RESULTS: There was minimal localization of 68Ga-labeled FAP-α inhibitor in the shoulders at day 0 (before surgery). There was significantly increased uptake in the shoulders with surgery compared with the contralateral sides without surgery at 3, 7, and 14 days. 68Ga-labeled FAP-α inhibitor uptake in the surgically treated shoulders increased gradually and peaked at 14 days followed by a decrease at 28 days. Gene expression for smooth muscle alpha (α)-2 (acta2), FAP-α, and fibronectin increased postsurgery followed by a drop at 28 days. Immunohistochemical analysis showed that FAP-α-positive cell density followed a similar temporal trend, peaking at 14 days. All trends matched closely with the PET/CT results. Biomechanical testing demonstrated a gradual increase in failure load during the healing process. CONCLUSIONS: 68Ga-labeled FAP-α inhibitor PET/CT allows facile, high-contrast in vivo 3-dimensional imaging of fibroblastic activity in a mouse rotator cuff repair model. CLINICAL RELEVANCE: Noninvasive imaging of activated fibroblasts using labeled radiotracers may be a valuable tool to follow the progression of healing at the bone-tendon interface.

Rotator cuff repair with biological graft augmentation causes adverse tissue outcomes.

Background and purpose - Biological patches can be used to augment rotator cuff tendon repair in an attempt to improve healing and reduce rates of re-rupture. However, little is known about the in vivo tissue response to these patches. We assessed native rotator cuff tissue response after surgical repair and augmentation with 2 commercially available extracellular matrix (ECM) patches. Patients and methods - Patients underwent a rotator cuff repair augmented with either GraftJacket (Wright Medical), Permacol (Zimmer Biomet), or no patch (Control), applied using an onlay technique. A sample of supraspinatus tendon was collected intraoperatively and 4 weeks post-surgery, using ultrasound-guided biopsy. Histology and immunohistochemistry were performed on all samples. Results - The Permacol group (n = 3) and GraftJacket group (n = 4) demonstrated some changes in native tendon ECM compared with the control group (n = 3). Significant disruption of the extracellular matrix of the repaired native supraspinatus, underlying both patches, was observed. The patches did not generally increase cellularity, foreign body giant cell count, or vascularity compared to the control group. 1 patient in the Permacol group had an adverse tissue immune response characterized by extensive infiltration of IRF5+, CD68+, and CD206+ cells, suggesting involvement of macrophages with a pro-inflammatory phenotype. No significant differences in protein expression of CD4, CD45, CD68, CD206, BMP7, IRF5, TGFß, and PDPN were observed among the groups. Interpretation - Histological and immunohistochemical analysis of native tendon tissue after patch augmentation in rotator cuff repair raises some concerns about a lack of benefit and potential for harm from these materials.

Microdialysis to Quantify Inflammatory Cytokines in the Glenohumeral Joint: A Brief Methods Report.

Microdialysis quantifies in vivo soft-tissue biochemical concentrations via passive diffusion of interstitial molecules through a porous membrane into a dialysate. The purpose of this pilot study was to evaluate a technique to measure inflammatory cytokines associated with rotator cuff tendinopathy by inserting a microdialysis catheter into the posterior glenohumeral joint. The technique was tested in a convenience sample of six pain-free, able-bodied veterans. Complete dialysate samples were collected in two participants. Two participants' sample volumes were smaller than what was required for analysis (30 µl) and thus were diluted. Catheter failures in two participants prevented collection altogether. Three cytokine concentrations were quantified: interleukin-1 receptor antagonist, interleukin 8, and regulated on activation, normal T-cell expressed and secreted. Microdialysis is not recommended for use in the glenohumeral joint, yet quantification of glenohumeral joint cytokines could yield valuable information to better understand pathophysiology of the joint and its surrounding tissues. Another technique, such as joint lavage, may be a more attractive alternative to overcome the limitations of microdialysis in the glenohumeral joint.

Increased interleukin 1ß levels in the subacromial fluid in diabetic patients with rotator cuff lesions compared with nondiabetic patients.

BACKGROUND: Diabetic patients have a high prevalence of shoulder pain and stiffness. Interleukin (IL) 1ß was reportedly correlated with shoulder stiffness and decreased shoulder function. We retrospectively compared the expression of IL-1ß in the subacromial synovial fluid between diabetic and nondiabetic patients with rotator cuff tearing. MATERIALS AND METHODS: We enrolled 68 patients with rotator cuff tearing (23 diabetic patients and 45 nondiabetic patients). The preoperative sum of range-of-motion deficit (SROMD), Constant score, and visual analog scale (VAS) score were obtained. Intraoperatively, subacromial synovial fluid was collected for the IL-1ß level measurement. Comparisons of IL-1ß levels, Constant scores, SROMD, and VAS scores between diabetic and nondiabetic patients were analyzed with the Mann-Whitney U test. RESULT: Diabetic patients with rotator cuff tearing had significantly increased subacromial IL-1ß levels (P = .048), SROMD (P < .001) and VAS scores (P = .022) and lower Constant scores (P < .001) than nondiabetic patients. The IL-1ß levels in the subacromial fluid were significantly correlated with the Constant score (r = -0.477, P < .001), VAS score (r = 0.698, P < .001), and SROMD (r = 0.293, P = .015) in all patients. CONCLUSION: The elevated IL-1ß levels in the subacromial fluid of patients with diabetes may explain the likelihood of pain and shoulder stiffness developing in these patients. We suggest more aggressive treatment for rotator cuff lesions in diabetic patients.

Implantation of a porcine acellular dermal graft in a primate model of rotator cuff repair.

BACKGROUND: Non-cross-linked xenogeneic extracellular matrix graft materials have typically elicited a hypersensitivity reaction when implanted into humans or other primates. The purpose of this study was to examine the histologic and immune response to a non-cross-linked porcine-derived dermal extracellular matrix graft processed to remove the α-gal epitope. MATERIALS AND METHODS: Eight African green monkeys were implanted with porcine acellular dermal matrix (Conexa Reconstructive Tissue Matrix; Tornier Inc, Edina, MN, USA) to repair and augment a partial excision defect of the supraspinatus tendon of the rotator cuff. Four animals each were sacrificed at 3 months and 6 months, and histologic samples were compared with tissues harvested from unoperated shoulders. RESULTS: Gross examination of grafted Conexa showed the appearance of integration proximally with tendon and distally with bone in each operated rotator cuff complex. Histologically, Conexa appeared to have remodeled to tendon-like architecture, with homogeneous distribution of fibroblast cells and parallel alignment of collagen fibers, with the direction of force evident by 3 months after implantation. Abundant vasculature observed at 3 months, which diminished to native tendon levels by 6 months, also indicated this to be a period of significant remodeling with an absence of significant inflammation, as evidenced by immunochemical methods and serum analysis. CONCLUSION: Conexa porcine acellular dermal matrix allows for incorporation of host tendon tissue without a hypersensitivity reaction in a primate model and should be a safe material for augmentation of human rotator cuff repair.

Interleukin-1ß stimulates stromal-derived factor-1α expression in human subacromial bursa.

Chemokines produced by synoviocytes of the subacromial bursa are up-regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF-1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL-1ß and IL-6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti-human antibodies to IL-1, IL-6, and SDF-1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL-1ß and IL-6) and SDF-1α expression was measured by ELISA and RT-PCR. SDF-1α, IL-1ß, and IL-6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose-dependent increase in SDF-1α production in the supernatants of cells treated with IL-1ß. SDF-1α mRNA expression was also increased in bursal cells treated with IL-1ß. IL-6 caused a minimal but not statistically significant increase in SDF-1α expression. SDF-1α, IL-1ß, and IL-6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF-1α gene expression and protein production are stimulated by IL-1ß. IL-1ß produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease.

Increased IL-1beta expression and myofibroblast recruitment in subacromial bursa is associated with rotator cuff lesions with shoulder stiffness.

We evaluated whether proinflammatory cytokine expression and myofibroblast recruitment in subacromial bursa was linked to rotator cuff lesions with shoulder stiffness. We analyzed expressions of IL-1beta, IL-6, and TNF-alpha in subacromial bursa and joint fluid collected from 14 patients with cuff tears with stiffness as a study group (Group I) and 14 patients with rotator cuff tears without shoulder stiffness as a control group (Group II) using real-time RT-PCR, immunohistochemistry, and ELISA. Myofibroblast apoptosis in subacromial bursa was analyzed using terminal deoxynucleotidyl transferase -mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) and alpha-smooth muscle actin immunofluorescence staining. Shoulder function was evaluated using the Constant score. Group I had higher mRNA expression (p < 0.001) and immunoreactivities (p < 0.001) of IL-1beta. They also had higher levels of IL-1beta, IL-6, and TNF-alpha in joint fluid. Increased IL-1beta mRNA expression in the subacromial bursa and IL-1beta levels in joint fluid were correlated with a preoperative deficit in shoulder motion (p < 0.001) and preoperative Constant scores (p < 0.001). Immunofluorescence observations showed that Group I subjects had more myofibroblasts (p < 0.001) than Group II. In Group II, a significant correlation was found between apoptotic myofibroblasts and total myofibroblasts (p = 0.002), but not in Group I (p = 0.510). Increased expression of IL-1beta and myofibroblast recruitment in the subacromial bursa in rotator cuff lesions are linked to shoulder stiffness.

Stromal cell-derived factor 1 (SDF-1, CXCL12) is increased in subacromial bursitis and downregulated by steroid and nonsteroidal anti-inflammatory agents.

Several studies have demonstrated that inflammation in the subacromial bursa is an important component in the pathogenesis of impingement syndrome. We have demonstrated in a previous study that many inflammatory cytokines, including stromal cell-derived factor 1 (SDF-1, CXCL12), are increased in the subacromial bursa [Blaine et al. 2005. J Shoulder Elbow Surg 14(Suppl 1):84S-89S]. SDF-1 is a potent chemotactic and angiogenic factor that stimulates recruitment of inflammatory cells. In the current study, we proposed that the resident cells in subacromial bursal tissue produce SDF-1, which can play a role in the inflammatory reponse of bursal tissue, and that this chemokine can be regulated by steroid (dexamethasone) and nonsteroidal anti-inflammatory medications (NSAIDs). Twenty-two subacromial bursa tissues (18 bursitis and 4 normal bursa) were obtained intraoperatively from patients during shoulder surgery and analyzed using the cDNA Array technique in accordance with an IRB approved protocol. cDNA array results were confirmed with real-time reverse transcription-polymerase chain reaction (RT-PCR). Bursal cells (from 4 normal bursa, 3 bursitis) and two normal bone marrow with whole tissue explants were cultured for one passage. Cell culture supernatants were collected and SDF-1 protein was detected with enzyme-linked immunosorbent assay (ELISA). Cultured bursal cells were treated with a COX-2 inhibitor and dexamethasone, and cells was harvested at 1-day and 4-day intervals. SDF-1 expression was evaluated by real-time RT-PCR and ELISA. cDNA Array analysis demonstrated that the gene expression of SDF-1 was increased in patients with subacromial bursitis compared to controls (p < 0.05). Real-time RT-PCR also revealed that the mRNA expression of SDF-1 in bursitis tissue is increased 10-fold over control tissue. While the normal bursal cells produced negligible amounts of SDF-1 protein, cultured cells derived from bursitis lesion released as much SDF-1 protein (235 pg/100,000 cells) as normal bone marrow stromal cells (283 pg/100,000 cells) as measured by ELISA. The addition of a COX-2 inhibitor and dexamethasone to bursitis cell lines led to decreased SDF-1 expression levels compared to untreated bursitis cell lines. These studies demonstrate that there is a significant elevation of SDF-1 expression in the subacromial bursa of patients with rotator cuff disease. Furthermore, this chemokine can be downregulated by COX-2 inhibitors and steroids. These results provide biologic evidence for the use of steroid and NSAIDs in the treatment of subacromial bursitis. In the future, targeted inhibition of molecules such as SDF-1 in the subacromial bursa may present a therapeutic strategy that may avoid the side effects of these other (steroid and NSAID) medications.

Interleukin-1-induced glenohumeral synovitis and shoulder pain in rotator cuff diseases.

Synovitis of the subacromial bursa has been identified as a main source of shoulder pain in rotator cuff diseases. Little interest, however, has been paid into the synovitis of glenohumeral joint. The mRNA expression levels of interleukin-1beta (IL-1beta) and interleukin-1 receptor antagonists produced in the synovitis reflect the magnitude of inflammation. The present study was undertaken to determine the relationship between mRNA expression levels of IL-1beta and its receptor antagonists (secreted interleukin-1 receptor antagonist (IL-1ra) and intracellular IL-1ra) in the synovium of the glenohumeral joint and shoulder pain in rotator cuff diseases, analyzing the synovial specimens by reverse transcriptase polymerase chain reaction. Thirty-five patients with rotator cuff diseases were candidates. Based on the presence of cuff perforation, they were divided into two categories: 16 with non-perforating tears and 19 with perforating tears. The degree of shoulder pain was evaluated by use of a visual analogue scale. The pain degree of non-perforating tears was significantly greater than that of perforating tears (P < 0.01). In contrast, the expression levels of the cytokine-mRNAs were constitutively greater in perforating tears than in non-perforating tears (P < 0.01, respectively). The expression levels of the cytokine-mRNAs were inversely correlated with the degree of pain (IL-1beta: r = 0.930; secreted IL-1ra: r = 0.861; intracellular IL-1ra: r = 0.932, P < 0.001 respectively). These results suggest that the expression levels of the cytokine-mRNAs in the synovium of the glenohumeral joint contribute less to the generation of shoulder pain in rotator cuff diseases.