ObjectiveTo investigate the effect of cytoskeleton modification on the adipogenic 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. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor γ (PPARγ), 1ipoprotein lipase (LPL), and fatty acid binding protein (aP2). ResultsThe final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P<0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P<0.05). ConclusionInhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.
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.
Objective To review the research progress of cell-scaffold complex in the tendon tissue engineering. Methods Recent literature concerning cell-scaffold complex in the tendon tissue engineering was reviewed, the research situation of the cell-scaffold complex was elaborated in the aspects of seed cells, scaffolds, cell culture, and application. Results In tendon tissue engineering, a cell-scaffold complex is built by appropriate seed cells and engineered scaffolds. Experiments showed that modified seed cells had better therapeutic effects. Further, scaffold functionality could be improved through surface modification, growth factor cure, mechanical stimulation, and contact guidance. Among these methods, mechanical stimulation revealed the most significant results in promoting cell proliferation and function. Through a variety of defect models, it is demonstrated that the use of cell-scaffold complex could achieve satisfactory results for tendon regeneration. Conclusion The cell-scaffold complex for tendon tissue engineering is a popular research topic. Although it has not yet met the requirement of clinical use, it has broad application prospects.
Objective To investigate the feasibility and effect of human amniotic membrane in prevention of tendon adhension after tendon sheat defect repair. Methods The amniotic membrane in size of 1.5 cm × 1.0 cm was harvested from human placenta which was voluntary donated from maternal after cesarean. Forty healthy male Leghorn chicken (aged 3-6 months) were selected, weighing (1.86 ± 0.04) kg. The model of flexor digitorum profundus tendon and tendon sheath defects was established at the third toe. After repair of the flexor digitorum profundus tendon, the human amniotic membrane was used to repair the tendon sheath defect in the right foot (group A), but tendon sheath defect was not repaired in the left foot (group B) . At 1, 2, 4, and 6 weeks after operation, the gross and histological observations were done; the degree of tendon adhesions was graded according to Tang’s tendon adhesion general observation grading standards; and the biomechanical properties (tendon slip length and total flexion angle) were tested. Results All animals survived after operation and incisions healed. Gross and histological observations showed that the new tendon sheath formed with time passing after operation in groups A and B; new tendon sheath was more maturer and smoother in group A than in group B. The degree of tendon adhesions in group A was significantly less than that in group B (P lt; 0.05) at 1 and 6 weeks after operation. The biomechanical test results showed there was no significant difference in the tendon slip length between 2 groups at 1 and 2 weeks after operation (P gt; 0.05), but the tendon slip length of group A was significantly longer than that of group B at 4 and 6 weeks after operation (P lt; 0.05). The total flexion angle of group A was significantly smaller than that of group B at 1, 2, 4, and 6 weeks after operation (P lt; 0.05). Conclusion It is effective in the prevention of tendon adhesion to use the amniotic membrane for repairing the tendon sheath defect, which is beneficial to recovery of the tendon sliding function.
Objective To investigate the effectiveness of the terminal extensor tendon reconstrution by palmaris longus tendon graft for the treatment of old mallet finger deformity. Methods Between February 2009 and February 2011, 32 patients with old mallet finger deformity were treated with palmaris longus tendon graft. There were 28 males and 4 females with an average age of 32.5 years (range, 22-58 years). The injury causes included sports injury in 26 cases and punctured injury in 6 cases. The index finger was involved in 8 cases, the middle finger in 3 cases, the ring finger in 16 cases, and the little finger in 5 cases. According to the Rockwell’s classification, all patients were classified as type I. The time from injury to operation was 4-16 weeks (mean, 6 weeks). Results Primary healing was obtained in all incisions; no necrosis, infection, or nail bed injury occurred. All patients were followed up 12-20 months (mean, 14 months). The patients had no pain or paresthesia of volar finger. According to Patel’s functional assessment system, the results were excellent in 8 cases, good in 21 cases, fair in 2 cases, and poor in 1 case at last follow-up, with an excellent and good rate of 90.6%. Conclusion Reconstruction of the terminal extensor tendon by palmaris longus tendon graft is a reliable method to treat old mallet finger deformity.
Objective To investigate the effect of canine decellularized tendon slices (DTSs) on tendon-bone healing in repairing rotator cuff injury of rabbit. Methods Canine DTSs were prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours from the adult Beagles Achilles tendons. Histological observation and cytocompatibility evaluation for the canine DTSs were performed in vitro. Twenty-four mature male New Zealand white rabbits, weighing 2.5-3.0 kg, were randomly selected. U-shaped defect of more than 50% of normal tendon in width and 8 mm in length was made in infraspinatus tendons of unilateral limb as the experimental group; the canine DTSs were used to repair defect, and the insertion of infraspinatus tendon on greater tuberosity of humerus was reconstructed in the experimental group. No treatment was done on the contralateral limb as the control group. At 4, 8, and 12 weeks after operation, the specimens were harvested for histological observation and biomechanical test. Results Histological examination showed that collagen fibers of canine DTSs were well preserved, without residual cells. The cytocompatibility examination showed that fibroblasts attached well to canine DTSs. Biomechanical test showed that the maximum load and stiffness increased significantly with time, and the maximum load and stiffness at 12 weeks were significantly higher than those at 4 and 8 weeks (P lt; 0.05). The maximum load and stiffness of the experimental group at 4 and 8 weeks were significantly lower than those of the control group (P lt; 0.05). The stiffness of the experimental group at 12 weeks was significantly lower than that of the control group (t= — 5.679, P=0.000), but no significant difference was found in the maximum load at 12 weeks between 2 groups (t=0.969, P=0.361). Histological observation showed that the control group displayed a 4-layer structure of the tendon-bone insertion. In the experimental group at 4 weeks, the tendon-bone interface was filled with granulation tissue, and a small amount of Sharpey’s fibers-like connected the tendon to bone; granulation tissue disappeared, and fibroblasts, Sharpey’s fiber, new cartilage, and chondrocytes significantly increased with time; tendon-bone interface became mature, but the tide line was not observed between the unmineralized fibrocartilage and mineralized fibrocartilage. Conclusion Canine DTSs prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours, can enhance the healing of host tendon-bone and improve the biomechanical characteristics of the rabbit infraspinatus tendon.
Objective To investigate the effectiveness of modified percutaneous suture in repairing acute closed Achilles tendon rupture by comparing with conventional open suture. Methods Between January 2006 and October 2009, 50 patients with acute closed Achilles tendon rupture were treated with modified percutaneous suture by making 5 small incisions at both sides of Achilles tendon and zigzag suture (improved group, n=22) and with Kessler suture (conventional group, n=28), respectively. No significant difference was found in gender, age, time from injury to operation between 2 groups (P gt; 0.05). Results In improved group, the patients achieved healing of incisions by first intention after operation and nocomplication occurred; however, incision infection occurred in 1 case, Achilles tendon re-rupture in 1 case, and incision scar contracture in 2 cases in conventional group. The operation time of improved group [(38.7 ± 6.6) minutes] was significantly shorter (t= —12.29, P=0.00) than that of conventional group [(52.3 ± 6.9) minutes]; the blood loss of improved group [(4.9 ± 2.0) mL] was significantly less (t= —25.20, P=0.00) than that of conventional group [(40.7 ± 7.1) mL]. The patients were followed up 2-3 years (mean, 29.9 months). The American Orthopaedic Foot and Ankle Society (AOFAS) score was 99.6 ± 1.0 in improved group and was 98.4 ± 3.0 in conventional group, showing no significant difference between 2 groups (t=1.66, P=0.10). Conclusion Comparison with conventional open suture, modified percutaneous suture has some advantages, such as easy operation, less complications, rapid recovery of limb function, and so on. Modified percutaneous suture is one of the best choices for the treatment of acute closed Achilles tendon rupture.
Objective Tri ptol ide can suppress immunological rejection reaction. To investigate the effect of tri ptol ide on allogenic tendon transplantation in repairing tendon defect in chicken. Methods The defect model of the third toes tendon was establ ished in 64 healthy-cleaning male Leghorn chickens (4-month-old, weighing 1.9-2.3 kg), which underwent allogenic tendon transplantation for repairing and were divided into 2 groups randomly (n=32). Tri ptol ide feeding[100 μg/(kg·d)] was given for 3 weeks in the experimental group and normal feeding in the control group. General condition of the chickens was observed after operation. The transplanted tendons were harvested from 4 chickens in each group for gross observation at 1, 2, 3, and 4 weeks after operation; the histological observation was performed at 1 and 3 weeks, and transmission electron microscope observation at 2 and 4 weeks. The blood and tendon were harvested from another 8 chickens in each group for flow cytometry and biomechanical tests respectively at 3 and 6 weeks. Results All chickens survived to the experiment end. Gross observation: with time extending, hyperemia and edema around transplanted tendon were rel ieved. Rarefaction adhering zone was seen in experimental group, and pyknotic adhering zone in control group. Histological observation: inflammatory reaction in experimental group was sl ighter than that in control group at 1 and 3 weeks. Transmission electron microscope observation: at 2 and 4 weeks, fibroblasts had big cell nucleus, more euchromatin, and l ittle heterochromatin in experimental group; however, there were small amount of rough endocytoplasmic reticulums with gentle expanded capsular space in control group, which contained sparse content. Flow cytometry test: at 3 and 6 weeks, peri pheral blood contained less CD4+ and CD8+ T lymphocytes in experimental group than in control group, and the ratio of CD4+ to CD8+ T lymphocyte significantly decreased in experimental group when compared with control group (P lt; 0.05). Biomechanical examination: at 3and 6 weeks, the maximum tensile strength in experimental group was bigger than that in control group, and tensile adhesion power in experimental group was smaller than that in control group. There were significant differences in the indexes between 2 groups (P lt; 0.05). Conclusion Tri ptol ide can suppress immunological rejection reaction, strengthen tendon healing strength, and reduce tendon adhesion in allogenic tendon transplantation.
Objective To review the appl ication of electrospinning in preparation of tendon tissue engineered scaffolds, to describe its appl ication effect and prospects. Methods Recent l iterature was extensively reviewed and summarized from various aspects, concerning the appl ication of electrospinning in preparing tendon tissue engineered scaffolds. Results Because of its huge surface and high porosity, the electrospun fibers prepared by electrospinning technology have been widely used in the manufacture of tendon tissue engineered scaffolds in recent years. A variety of materials, including polylactic acid, have been successfully electrospun into various types of tendon tissue engineered scaffolds, and goodresults in the repair of tendon defect were achieved. Conclusion The electrospinning technology has provide a new way for the preparation of the tendon tissue engineered scaffolds, with the perfection of the technology they will have broad application prospects in the field of tendon tissue engineering.
Objective To study the effectiveness of anterior cruciate l igament (ACL) reconstruction using autologous periosteum wrapping tendon allograft by comparing with using simple tendon allograft. Methods Between March 2008 and November 2008, 68 patients with ACL injury were treated, who were in accordance with the inclusion criteria. They were divided into 2 groups randomly according to different treatment methods: ACL was reconstructed with autologous periosteum wrapping tendon allograft in 31 patients (test group) and with simple tendon allograft (control group) in 37 patients. There was no significant difference in gender, age, disease duration, the cause of injury, and functional score preoperatively between 2 groups (P gt; 0.05). Anatomic single-bundle ACL reconstruction was performed in 2 groups. Results Little exudation at tibial tunnel incision was found in 1 case respectively in both groups at 2 weeks after operation and was cured by dressing change and antibiotics. The other incisions healed by first intention. The patients were followed up 24-29 months (mean, 26 months) in the test group and 24-32 months (mean, 27 months) in the control group. CT showed bone tunnel enlargement in both groups at 2 years after operation, but the rate of the tunnel enlargement was less inthe test group (5/31, 16.1%) than in the control group (14/37, 37.8%), showing significant difference (χ2=3.948, P=0.047). At 2 years after operation, the results of Lachman test and pivot shift test were negative in 23 cases (74.2%) and 25 cases (80.6%) of the test group, and in 26 cases (70.3%) and 30 cases (81.1%) of the control group, respectively. KT-1000 examination showed the displacement of the test group [(1.74 ± 0.88) mm] was less than that of the control group [(2.36 ± 0.83) mm], showing significant difference (t= —2.979, P=0.004). There was no significant difference in Lysholm score, Hospital for Special Surgery (HSS) score, Tegner score, and International Knee Documentation Committee (IKDC) score between 2 groups at 2 years after operation (P gt; 0.05). Conclusion Compared with simple tendon allograft, ACL reconstruction with autologous periosteum wrapping tendon allograft can improve tendon-bone heal ing, and decrease the rate of bone tunnel enlargement, so it has good short-term outcome.