Objective To investigate the effects of bone morphogenetic protein 2 (BMP-2) on the chondrogenic differentiation of human Achilles tendon-derived stem cells (hATDSCs) in vitro. Methods Achilles tendon was harvested from a voluntary donor with acute Achilles tendon rupture. And nucleated cells were obtained by digesting with collagenase and were cultured to the 3rd passage. The flow cytometry was used to measure the immunophenotyping; and Oil red O staining, alizarin red staining, and Safranin O/fast green staining were used to identify the adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation, respectively. The hATDSCs pellet was cultured in complete culture medium with (experimental group) or without recombinant human BMP-2 (rhBMP-2) (control grup) for 3 weeks. Chondrogenic differentiation of hATDSCs was evaluated by HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II; and the mRNA expressions of SOX9, collagen type II, and Aggrecan were detected by real-time fluorescence quantitative PCR. Results Primary hATDSCs cultured in vitro showed clonal growth; after cell passage, homogeneous spindle fibroblast-like cells were seen. The cells were positive for CD44, CD90, and CD105, while negative for CD34, CD45, and CD146. The results were positive for Oil red O staining at 3 weeks after adipogenic differentiation, for alizarin red staining at 4 weeks after osteogenic differentiation, and for Safranin O/fast green staining at 3 weeks after chondrogenic differentiation. After hATDSCs were induced with rhBMP-2 for 3 weeks, pellets formed in the experimental group, and the size of pellets was significantly larger than that in the control group; the results of HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II were all positive. The results of real-time fluorescence quantitative PCR showed that the mRNA expressions of SOX9, collagen type II, and Aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion BMP-2 can promote proteoglycan deposition and induce chondrogenic differentiation of hATDSCs in vitro. The effect of BMP-2 on hATDSCs might provide a possible explanation for histopathological changes of tendinopathy.
【Abstract】 Objective Prostaglandin E2 (PGE2) production increases in human tendon fibroblasts after the tendon injuries and repetitive mechanical loading in vitro. To analyze the relations between PGE2 and tendinopathy by observing the changes of collagen content and proportion after the Achilles tendon of rabbits is repeatedly exposed to PGE2. Methods Twenty-four Japanese rabbits (aged 3-4 months, weighing 2.0-2.5 kg, and male or female) were equally randomized into 2 groups according to injection dose of PGE2: low dose group (50 ng) and high dose group (500 ng). Corresponding PGE2 (0.2 mL) was injected into the middle segment of the Achilles tendon of hindlimb, the same dose saline into the same site of the other side as controls once a week for 4 weeks or 8 weeks. The Achilles tendons were harvested at 4 and 8 weeks after injection. HE staining was used to observe the cell structure and matrix, and picric acid-sirius red staining to observe the distribution and types of collagen fibers, and transmission electron microscopy was used to measure the density of the unit area and diameter of collagen fibers. Results HE staining showed that collagen structural damage was observed in low dose and high dose groups. Picric acid-sirius red staining showed that the content of type I collagen significantly decreased while the content of type III collagen significantly increased in experimental side of 2 groups at 4 and 8 weeks after injection when compared with control sides (P lt; 0.05). The content of type I collagen was significantly lower and the content of type III collagen and ratio of type III to type I were significantly higher in high dose group than in low dose group (P lt; 0.05). Transmission electron microscopy showed that the collagen fibers density of unit area was significantly lower and the diameter was significantly smaller in high dose and low dose groups than in the controls (P lt; 0.05), and in high dose group than in low dose group (P lt; 0.05). Conclusion Repeat exposure of the Achilles tendon of rabbit to PGE2 can cause the decrease of type I collagen, the increase of type III collagen, the reverse ratio of type I to type III, reduced unit density of collagen fibers, and thinner collagen fibers diameter, which is related with tendinopathy.
ObjectiveTo investigate the effect of different mechanical tensions on the expressions of RhoA/Rho associated protein kinases (ROCK) in rat tendon stem cells (TSCs). MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged, 2-3 months; weighing, 200-250 g) by enzymatic digestion method and cultured for 2-3 passages, then seeded on micro groovdishes. The 4% (4% stretch group) and 8% (8% stretch group) mechanical stretching was performed for 4 hours every day at 1 Hz. After 1, 2, and 3 days, the protein and mRNA expressions of RhoA and ROCK were measured by Western blot and real-time quantitative PCR. The cell proliferation was measured by cell counting kit 8. The cells were not stretched as control group. ResultsThe TSCs at passage 2 showed a cobble-stone shape and aggregation growth; TSCs seeded on micro groovdishes showed random growth, and the cells grew along the stretching direction after mechanical stretching. The mRNA expressions of RhoA and ROCK in control group, 4%, and 8% stretch groups showed an increasing tendency at 1, 2, and 3 days, showing significant difference between groups (P<0.05). The protein expressions of RhoA and ROCK in 4% and 8% stretch groups were similar to those in control group at 1 day (P>0.05), but the expressions in 4% and 8% stretch groups showed an increasing tendency at 2 and 3 days, which were significantly higher than those in control group (P<0.05). The cell proliferation of 8% stretch group was significantly lower than that of 4% stretch group and control group at each time point (P<0.05), but no significant difference was found between 4% stretch group and control group (P>0.05). ConclusionThe expressions of RhoA and ROCK of rat TSCs are positively correlated with stretch intensity. So RhoA/ROCK may be an important molecule in TSCs after mechanical stretching.
ObjectiveTo evaluate the effectiveness of the combined tenodesis for proximal lesions of biceps tendon with massive rotator cuff tear by arthroscopy. MethodsBetween January 2011 and June 2013, 48 patients with massive rotator cuff tear and proximal lesions of biceps tendon underwent combined tenodesis under arthroscopy, and the clinical data were retrospectively analyzed. Of 48 cases, 22 were male and 26 were female with an average age of 46 years (range, 35-59 years); 12 cases had clear history of trauma. The disease duration ranged from 1 to 57 months (mean, 4.6 months). All cases suffered from moderate to severe shoulder pain, the strength and the range of motion (ROM) declined when compared with those of the other side. According to Goutallier classification standard, 3 cases were rated as grade 0, 18 cases as grade 1, and 27 cases as grade 2. The operation time and complication were recorded. The visual analogue scale (VAS) score, ROM, the strength of flexed elevation and elbow flexion, Constant-Murley score, University of California Los Angeles (UCLA) score, American Shoulder and Elbow Surgeons (ASES) score, Mayo elbow performance score (MEPS) were used to evaluate the effectiveness. ResultsThe operations were performed successfully, and incisions healed primarily. The operation time was 120-160 minutes (mean, 135 minutes). One case had shoulder joint swelling with wound bleeding, which was cured after proper treatment. All 48 patients were followed up 12-18 months (mean, 13.9 months). The results of MRI showed good healing of tendon at 6 months after operation. When compared with preoperative values, VAS score was significantly decreased (P<0.05); the ROM in forward flexion, external rotation, and internal rotation, and the strength of flexed elevation were significantly increased (P<0.05); and ASES score, Constant-Murley score, and UCLA score were significantly improved at 12 months after operation (P<0.05). No significant difference was observed in MEPS score and elbow flexion strength (P>0.05). ConclusionThe technique of combined tenodesis under arthroscopy can obtain satisfactory clinical outcomes in treating proximal lesions of biceps tendon with massive rotator cuff tear.
ObjectiveTo investigate the effect of transforming growth factor β3 (TGF-β3) at different concentrations on the differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated with TGF-β3 at the concentrations of 5.0, 2.5, 1.0, and 0 ng/mL. At 1, 3, and 5 days, the mRNA expressions of tendogenic differentiation related genes[collagen type Ⅰ,tenascin C (TNC), tenomodulin (TNMD), scleraxis (Scx)], osteogenic differentiation related genes[Runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP)], chondrogenic differentiation genes (Sox9 and collagen type Ⅱ),and adipogenic differentiation genes[AP2 and peroxisome proliferator-activated receptor γ (PPARγ)] were measured by real-time quantitative PCR (qRT-PCR). ResultsTSCs could differentiated in different directions after treated with TGF-β3 at different concentrations at different time points. TGF-β3 was able to induce TSCs differentiated into tenocytes, which was related to the concentration and time of duration, and the two factors have interaction. Stimulation of TGF-β3 at low concentration and for short time could inhibit non-tendogenic differentiation of TSCs, but at high concentration and for long time, TGF-β3 enhanced TSCs differented into osteocytes or chondrocytes. ConclusionEffects of TGF-β3 on TSCs differentiation are complicated and depend on the concentration and time of duration, which may be a key factor between tendogenic and non-tendogenic differentiations of TSCs.
Objective To explore the effect of different intensity treadmill training on the repair of micro-injured Achilles tendon induced by collagenase in rats. Methods Seventy-two 8-week-old male Sprague Dawley rats (weighing, 200-250 g) were selected. After adaptive treadmill training for 1 week, rats were injected with 30 μL type I collagenase solution (10 mg/mL) into both Achilles tendons to make micro-injured Achilles tendon models. After 1 week of cage feeding, the rats were randomly divided into 3 groups: the control group, the low-intensity group, and the high-intensity group, 24 rats each group. The rats in control group could move freely, and the rats underwent daily treadmill training at the intensity of 13 m/min and 20 min/d in the low-intensity group and at the intensity of 17 m/min and 60 min/d in the high-intensity group. At immediate, 1 week, and 4 weeks after training, bilateral Achilles tendons were collected from 8 rats of each group for gross observation, histological analysis, and mechanical testing. Results At immediate after training, there was no significant difference in the gross observation, histological observation, and biomechanical properties of the Achilles tendon between groups (P>0.05). The gross observation showed connective tissue hyperplasia near Achilles tendon and lackluster tendon in each group at 1 week; hyperplasia significantly reduced in the low-intensity group when compared with the control group, and there were more connective tissue and a large number of neovascularization in the high-intensity group at 4 weeks. At 1 week, there was no significant difference in the semi-quantitative histological total score between groups (P>0.05), but there were significant differences in vascularity between low-intensity group or high-intensity group and control group (P<0.05). At 4 weeks, the semi-quantitative histological total score was significantly higher in high-intensity group than control group and low-intensity group (P<0.05), and in control group than low-intensity group (P<0.05). There were significant differences in collagen arrangement, cell morphology, abnormal cells, and vascularity between low-intensity group and high-intensity group or control group (P<0.05). And there was significant difference in abnormal cells between high-intensity group and control group (P<0.05). The mechanical testing showed that there was no significant difference in cross-sectional area of the Achilles tendon, the ultimate force, tensile strength, and elastic modulus between groups at 1 week (P>0.05); the low-intensity group was significantly higher than the control group in the ultimate force and the tensile strength (P<0.05), and than high-intensity group in the ultimate force and elastic modulus (P<0.05), but no significant difference was found in the other indexes between groups (P>0.05) at 4 weeks. Conclusion Low-intensity treadmill training can promote the repair of rat micro-injured Achilles tendon induced by collagenase.
ObjectivesTo systematically review the clinical efficacy of ultrasound-guided percutaneous lavage (UGPL) for calcifying tendinitis of rotator cuff.MethodsPubMed, The Cochrane Library, EMbase, WanFang Data, CBM, VIP and CNKI databases were electronically searched to collect the randomized controlled trials (RCTs) of ultrasound-guided percutaneous irrigation for calcifying tendinitis of rotator cuff from inception to September 2017. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies. Meta-analysis was performed by using RevMan 5.3 software.ResultsA total of 7 RCTs involving 587 patients were included. The results of meta-analysis showed that: there were significant differences between the UGPL group and extracorporeal shock wave therapy (ESWT) group in 12-month follow-up visual analogue scale (VAS) (MD=–1.96, 95%CI –2.18 to –1.75, P<0.000 01), the average particle size of the calcium deposition (MD=–3.13, 95%CI –5.05 to –1.22,P<0.001), the calcification disappearance rate (RR=1.65, 95%CI 1.36 to 2.01,P<0.000 01). However, there were no significant differences between the two groups in 6-week follow-up VAS (MD=–0.85, 95%CI –2.84 to 1.14,P=0.40), and the complications (RR=1.20, 95%CI 0.03 to 49.69, P=0.93).ConclusionsCurrent evidence shows that UGPL has certain clinical effect for the calcifying tendinitis of rotator cuff. Compared with ESWT, UGPL may be superior in clinical pain relief and calcification clearance. Due to limited quality and quantity of the included studies, more high quality studies are required to verify above conclusions.