Masafumi Gotoh

Hyaluronic acid inhibits mRNA expression of proinflammatory cytokines and cyclooxygenase-2/prostaglandin E2 production via CD44 in interleukin-1-stimulated subacromial synovial fibroblasts from patients with rotator cuff disease

A growing body of evidence supports use of intraarticular hyaluronic acid (HA) injection in patients with rotator cuff disease. However, the mechanism of its anti‐inflammatory action has not been clarified. We examined the effects of HA on the expression of mRNAs for proinflammatory cytokines (IL‐1β, IL‐6, and TNF‐α and COX‐2/PGE2 production in IL‐1‐stimulated subacromial‐synovium fibroblasts (SSF) derived from patients with rotator cuff disease. Various concentrations of HA were added to monolayer SSF cultures in the presence of IL‐1β. Gene expression levels were analyzed by quantitative real‐time reverse transcription‐polymerase chain reaction. Intracellular production of COX‐2 was identified by Western blotting. PGE2 concentrations in the culture media were measured by ELISA. CD44 blocking with OS/37 was performed to investigate the mechanism of action of HA. Immunofluorescence cytochemistry confirmed binding of HA and the presence of CD44 on SSF. Exogenous HA significantly and dose‐dependently decreased expression of proinflammatory cytokine mRNAs and COX‐2/PGE2 production in IL‐1‐stimulated SSF. Pretreatment with OS/37 reversed the inhibitory effects of HA. These results provide a basis for explaining why HA is effective for the treatment of rotator cuff disease.

Vascular endothelial growth factor (VEGF) expression in the subacromial bursa is increased in patients with impingement syndrome

Vascular endothelial growth factor (VEGF), which is known to be an angiogenetic factor, plays an important role in the inflammation of synovial tissue. To investigate the relationships between VEGF and clinical symptoms in rotator cuff disease, VEGF expression was examined using RT‐‐PCR and immunohistochemical analysis in 50 patients with this disease (26 with full‐thickness cuff tear, 12 with partial‐thickness tear, and 12 with subacromial bursitis). VEGF mRNA expression was detected in 40 out of 50 patients by RT—PCR. VEGF mRNA expression was found more frequently in the patients with motion pain (39 out of 41) than in those without motion pain (1 out of 9) with statistical significance (Fishers test, P < 0.001). Thirty‐one out of 33 patients with synovial proliferation showed VEGF mRNA expression, whereas the expression of this transcript was found in 9 out of 17 patients without synovial proliferation. This association with synovial proliferation was also significant (Fishers test, P = 0.0013). Thirty out of 41 patients with motion pain had synovial proliferation but 3 out of 9 patients without motion pain had synovial proliferation. In all these 30 patients with both motion pain and synovial proliferation, VEGF mRNA expression was detected. This association between motion pain and synovial proliferation was also significant (Fishers test, P < 0.05). The mean vessel count and area in subacromial bursa expressing VEGF was significantly higher than in those without VEGF (Mann‐Whitneys U test, P < 0.01). These results suggested that VEGF expression is associated with vascularity, synovial proliferation and shoulder motion pain in the rotator cuff disease.

Collagen Production at the Edge of Ruptured Rotator Cuff Tendon is Correlated With Postoperative Cuff Integrity

PURPOSE The purpose was to evaluate the correlation between messenger RNA (mRNA) expression of collagen at the edge of the ruptured rotator cuff tendon and postoperative cuff integrity. METHODS The edge of the ruptured tendon was sampled during open rotator cuff surgery in 12 patients with full-thickness rotator cuff tears (mean age, 58.2 years). The mean period from symptom onset was 9.3 months (range, 1 to 36 months), and the mean tear size was 4.1 cm. As controls, rotator cuff tendons with no gross rupture were taken from 5 fresh cadavers. Production of type I and type III collagen was examined by real-time reverse transcription polymerase chain reaction. By use of magnetic resonance imaging, postoperative cuff integrity was evaluated based on the classification of Sugaya et al. and then scored, ranging from 5 points for type I to 1 point for type V. RESULTS Looking at the mRNA of type I and type III collagen in tendons, we found that the expression of mRNA for both collagen types in ruptured tendons was significantly greater than in control tendons (P = .0462 for type I collagen and P = .0306 for type III collagen). Correlating the mRNA of type I and type III collagen with repaired cuff integrity on postoperative magnetic resonance imaging, we found a close relation between expression of mRNA for both collagen types and postoperative rotator cuff integrity (r = 0.63 [P = .038] for type I collagen and r = 0.626 [P = .03] for type III collagen). Furthermore, expression of type I collagen mRNA showed a significant inverse correlation with the period from symptom onset (r = -0.845, P < .0005). CONCLUSIONS This study showed that expression of mRNA for type I and type III collagen at the edge of the ruptured rotator cuff tendon was significantly correlated with postoperative cuff integrity and that mRNA expression for type I collagen was significantly associated with the period from symptom onset. These results may suggest that conservative treatment should not be prolonged if patients do not respond within a certain period. LEVEL OF EVIDENCE Level III, prognostic case-control study.

Risk factors for shoulder re-dislocation after arthroscopic Bankart repair

BackgroundRecent studies have shown effective clinical results after arthroscopic Bankart repair (ABR) but have shown several risk factors for re-dislocation after surgery. We evaluated whether patients are at a risk for re-dislocation during the first year after ABR, examined the recurrence rate after ABR, and sought to identify new risk factors.MethodsWe performed ABR using bioabsorbable suture anchors in 102 consecutive shoulders (100 patients) with traumatic anterior shoulder instability. Average patient age and follow-up period was 25.7 (range, 14–40) years and 67.5 (range, 24.5–120) months, respectively. We evaluated re-dislocation after ABR using patient telephone interviews (follow-up rate, 100%) and correlated re-dislocation with several risk factors.ResultsRe-dislocation after ABR occurred in nine shoulders (8.8%), of which seven sustained re-injuries within the first year with the arm elevated at 90° and externally rotated at 90°. Of the remaining 93 shoulders without re-dislocation, 8 had re-injury under the same conditions within the first year. Thus, re-injury within the first year was a risk for re-dislocation after ABR (P < 0.001, chi-squared test). Using multivariate analysis, large Hill-Sachs lesions (odds ratio, 6.77, 95% CI, 1.24–53.6) and <4 suture anchors (odds ratio, 9.86, 95% CI, 2.00–76.4) were significant risk factors for re-dislocation after ABR.ConclusionsThe recurrence rate after ABR is not associated with the time elapsed and that repair strategies should augment the large humeral bone defect and use >3 anchors during ABR.

Hyaluronan modulates proliferation and migration of rabbit fibroblasts derived from flexor tendon epitenon and endotenon.

PURPOSE There is a growing body of evidence supporting the use of hyaluronan (HA) for treatment of injured tendons, although the mechanism of the healing effect has not yet been clarified. We therefore investigated the effects of HA on the proliferation and migration of tendon fibroblasts derived from rabbit flexor tendon epitenon and endotenon. METHODS From explanted rabbit intrasynovial flexor tendons (n = 5), we cultured tendon fibroblasts derived from the epitenon and endotenon. CD44 expression on the tendon fibroblasts was detected by flow cytometric analysis. Various concentrations of HA (0.1-5.0 mg/mL) were added to monolayer-cultured tendon fibroblasts. We evaluated cell proliferation by recording changes in cell number, and measured cell migration by wound-healing assay. RESULTS Flow cytometric analysis detected CD44 expression on the tendon fibroblasts. Treatment with HA at various concentrations notably and dose dependently inhibited cell proliferation and promoted cell migration. CONCLUSIONS Hyaluronan modulates the proliferation and migration of rabbit fibroblasts derived from the flexor tendon epitenon and endotenon.

Effects of Hyaluronan on Cell Proliferation and mRNA Expression of Procollagens α1 (I) and α1 (III) in Tendon-Derived Fibroblasts from Patients with Rotator Cuff Disease: An in Vitro Study

Background Hyaluronan (HA) improves postoperative recovery after flexor tendon surgery, preventing postoperative adhesion. However, its influence on the rotator cuff tendon after cuff repair has not yet been clarified in detail. Hypothesis Hyaluronan is likely to modulate cell proliferation and mRNA expression of procollagens α1 (I) and α1 (III) in tendon-derived fibroblasts in patients with rotator cuff disease. Study Design Controlled laboratory study. Methods The study subjects were 10 patients with rotator cuff disease, with an average age of 62 years (range, 44-72). Various concentrations of HA (1.0-5.0 mg/mL) were added to monolayer-cultured tendon-derived fibroblasts from these patients. Hyaluronan binding and CD44 expression on the tendon-derived fibroblasts were evaluated by confocal microscopy using fluorescein-conjugated HA and antihuman CD44 antibody (OS/37). Cell proliferation was evaluated by recording changes in cell number. The levels of expression of procollagen α1 (I) and α1 (III) mRNA were measured by real-time reverse transcriptase polymerase chain reaction. Results Immunofluorescence cytochemistry detected constitutive binding of HA and CD44 expression on the tendon-derived cells. Treatment with various concentrations of HA significantly inhibited cell proliferation and decreased the expression level of procollagen α1 (III) mRNA, but not that of procollagen α1 (I) mRNA, in the tendon-derived fibroblasts. Conclusion Hyaluronan modulates cell proliferation and the expression level of procollagen α1 (III) mRNA, but not that of procollagen α1 (I), in fibroblasts from patients with rotator cuff disease. Clinical Relevance Postoperative use of exogenous HA may allow the healing of a repaired rotator cuff tendon with minimal adhesion.

Vascular endothelial growth factor 121 and 165 in the subacromial bursa are involved in shoulder joint contracture in type II diabetics with rotator cuff disease

Vascular endothelial growth factor (VEGF) is a glycoprotein that plays an important role in neovascularization and increases vascular permeability. We reported that VEGF is involved in motion pain of patients with rotator cuff disease by causing synovial proliferation in the subacromial bursa (SAB). The present study investigates whether VEGF is also involved in the development of shoulder contracture in diabetics with rotator cuff disease. We examined 67 patients with rotator cuff disease, including 36 with complete cuff tears, 20 with incomplete tears, and 11 without apparent tears (subacromial bursitis). The patients were into groups according to the presence or absence of diabetes (14 type II diabetics and 53 non‐diabetics). Specimens of the synovium of the SAB were obtained from all patients during surgery. Expression of the VEGF gene in the synovium of the subacromial bursa was evaluated by using the reverse transcriptase polymerase chain reaction. The VEGF protein was localized by immunohistochemistry, and the number of vessels was evaluated based on CD34 immunoreactivity. The results showed that VEGF mRNA was expressed in significantly more diabetics (100%, 14/14) than in non‐diabetics (70%, 37/53) (P = 0.0159, Fishers test). Investigation of VEGF isoform expression revealed VEGF121 in all 14 diabetics and in 37 of the 53 non‐diabetics, VEGF 165 in 12 of the 14 diabetics and in 21 of the 53 non‐diabetics, and VEGF 189 in 1 of the 14 diabetics and in 2 of the 53 non‐diabetics. No VEGF206 was expressed in either group. VEGF protein was localized in both vascular endothelial cells and synovial lining cells. The mean number of VEGF‐positive vessels and the vessel area were also significantly greater in the diabetics (p < 0.015, Mann‐Whitney U test). Synovial proliferation and shoulder joint contracture were more common in the diabetics (P = 0.0329 and P = 0.073, respectively; Fishers test). The mean preoperative range of shoulder motion significantly differed in terms of elevation between two groups: 103.8° in diabetics and 124.9° in no diabetics (p = 0.0039 Mann–Whitney U test). In contrast, external rotation did not significantly differ: 44° in diabetics and 49° in non‐diabetics (p ° 0.4957, Mann–Whitney U test). These results suggest that VEGF121 and VEGF165 expression in the SAB is responsible for the development of shoulder joint contracture, especially in elevation, among type II diabetic patients with rotator cuff disease.

Hyperadiponectinemia enhances bone formation in mice

BackgroundThere is growing evidence that adiponectin, a physiologically active polypeptide secreted by adipocytes, controls not only adipose tissue but also bone metabolism. However, a role for adiponectin in bone development remains controversial.MethodsWe therefore investigated the endocrine effects of adiponectin on bone metabolism using 12-week-old male transgenic (Ad-Tg) mice with significant hyperadiponectinemia overexpressing human full-length adiponectin in the liver.ResultsIn Ad-Tg mice, the serum level of osteocalcin was significantly increased, but the levels of RANKL, osteoprotegerin, and TRAP5b were not. Bone mass was significantly greater in Ad-Tg mice with increased bone formation. In contrast, bone resorption parameters including the number of osteoclasts and eroded surface area did not differ between Ad-Tg and their littermates.ConclusionsThese findings demonstrate that hyperadiponectinemia enhances bone formation in mice.

Hyaluronan modulates cell proliferation and mRNA expression of adhesion-related procollagens and cytokines in glenohumeral synovial/capsular fibroblasts in adhesive capsulitis.

There is a growing body of evidence supporting the use of hyaluronan (HA) in patients with adhesive capsulitis of the shoulder, although the mechanisms of the effect have not yet been clarified. This in vitro study examined the effects of HA on glenohumeral synovial/capsular fibroblasts (GSCFs) from patients with adhesive capsulitis of the shoulder. The study subjects were seven patients with primary or secondary adhesive capsulitis of the shoulder (average age: 55 years; range: 42–65). Synovial/capsular specimens were obtained from the rotator interval of each patient during arthroscopy. Part of the tissue specimen was used for histological analysis. The remainder of the tissue was prepared for cell culture. Various concentrations of HA (0.0–4.0 mg/mL) were added to the monolayer‐cultured GSCFs from these patients. Histological analysis consistently demonstrated chronic nonspecific inflammation with synovial hyperplasia, proliferation of vessels and fibroblasts, and increased amount of extracellular matrix. Treatment with HA at various concentrations significantly and dose‐dependently inhibited cell proliferation and decreased the expression levels of mRNA for adhesion‐related procollagens and cytokines. Pretreatment with OS/37 did not reverse the inhibitory effect of HA. These results suggest that HA modulates cell proliferation and expression of the mRNA of adhesion‐related procollagens and cytokines in GSCFs, preventing the progression of adhesion formation in patients with adhesive capsulitis of the shoulder.

Interleukin-6-induced Activation of Signal Transducer and Activator of Transcription-3 in Ruptured Rotator Cuff Tendon:

The aim of this study was to examine interleukin-6 production and the activation of signal transducer and activator of transcription-3 (STAT3) in ruptured rotator cuff tendon. Specimens of ruptured rotator cuff tendons were analysed using real-time reverse transcriptase polymerase chain reaction, Western blotting and immunohistochemistry. Specimens of co-existing inflammatory subacromial synovia were examined for comparison. The level of interleukin-6 messenger RNA was increased in ruptured rotator cuff tendon as well as in subacromial synovium. Western blot analysis showed constitutive production of activated, phosphorylated STAT3 in ruptured rotator cuff tendon and co-existing subacromial synovium. Immunohistochemical examination detected cells producing interleukin-6, interleukin-6 receptor and phosphorylated STAT3 in ruptured rotator cuff tendon, mainly in proliferative vessels and, to a lesser extent, in tendon fibroblasts around the vessels. This study demonstrates that activation of STAT3 induced by interleukin-6 is promoted mainly by proliferative vessels in ruptured rotator cuff tendon.