Objective To review the research and appl ication of functional tissue engineered tendons (FTETs). Methods Recent l iterature concerning the research of FTETs was reviewed and analyzed. Results Functional tissue engineering (FTE) was a new approach that placed an emphasis on the importance of mechanical stress in determining the success of tissue engineered constructs and the effect of tissue remodel ing in vivo. The concept of FTE was introduecd into the research of tissue engineered tendons; by measuring in vivo loads of normal tendon and using the information as a guidel ine, theappropriate tissue engineered tendon which can withstand in vivo loads was designed. It would be possible to solve the problems that the biomechanical function of the tissue engineered tendons could not meet the requirements of the loading environment in vivo. Conclusion FTETs have a more promising future for the treatment of tendon defects.
Objective To explore a rapid histological preparation method to observe morphology and composition distribution of tendon collagen fascicle and endotendinum. Methods Taking porcine superflexor tendon of foot as an example, tendons were sliced into sections with 6 μm by frozen section technology, after which general observation of the section integrity was carried out. After fixed with 10% neutral buffered formalin and performed with HE staining, the tissue integrity and ice crystal formation were observed under microscope. Sections were then divided into 5 groups by different methods of dyeing. Group A: Priodic acid-Shiff (PAS) staining; group B: Masson staining; group C: reticular fibers staining; group D: immunohistochemical and immunofluorescent staining of type Ⅲ collagen; group E: the sections were baked at 65℃ for 10 minutes and stained with Masson. The composition distribution of tendon collagen fascicle and endotendinum in different groups were observed. Results From general observation, the frozen section of tendon tissue was complete and continuous. Although the tissue integrity in the tendon sections could be seen and no ice crystal was formed, the composition distribution could not be identified by HE staining. The entire tendons in groups A, B, and C were dyed, and the composition distribution of collagen fascicle and endotendinum could not be identified. The endotendinum in group D was stained weakly positive for type Ⅲ collagen alone, and the two components were differentiated dyed but the contrast was not obvious. In group E, the collagen fascicle and endotendinium were differentiated dyed and the two components in tendon tissue were clearly visible. Conclusion The morphology and the composition distribution of tendon collagen fascicle and endotendinum can be characterized rapidly and accurately, using a combination of baking at 65℃ for 10 minutes and Masson staining after porcine superflexor tendons were sliced by frozen section technology.
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 study the possibil ity of bone marrow mesenchymal stem cells (BMSCs) differentiation into tenocytes (TCs) under strain stimulation by co-culture of BMSCs-small intestinal submucosa (SIS) composites in vitro. Methods BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats. Inducing method of multiple differentiation and flow cytometry were appl ied to identify the cells. The stress-strain curve of SIS was measured with Instron machine. Purified BMSCs (2nd passage, 2.5 × 105 cells/cm2) were seeded on SIS (3 cm × 1 cm at size) and cultured for 2 daysand then continued for another 5 days under strain stimulation (stretching frequency was 0.02 Hz, action time was 15 minutes/ hour and 12 hours/day, strain ampl itude was 5%) as experimental group, while the BMSCs-SIS composites were sustained static culture as control group. TCs were isolated from tail of 1-week-old SD rats. TCs-SIS composites were cultured under non-strained as positive control group. Scanning electron microscope (SEM) was used to examine the morphological changes of BMSCs after strain stimulation. The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit. Results The BMSCs could be induced to differentiate into osteoblasts and l ipocytes, and showed the results of CD34-, CD45-, and CD90+, which were accorded with the biological characteristics of BMSCs. The failure test of SIS showed that the average elastic strain was 39.5%. SEM observation showed that the strain-stimulated BMSCs had the TCs-l ike morphological characteristics. The contents of Scleraxis and Tenomodul in in supernatant of experimental group, control group, and positive control group were (3.56 ± 0.91) μmol/L and (4.27 ± 1.10) μmol/L, (0.23 ± 0.14) μmol/L and (0.16 ± 0.10) μmol/L, and (14.73 ± 2.30) μmol/L and (10.65 ± 1.51) μmol/L, respectively. There were significant differences among 3 groups (P lt; 0.05). Conclusion Appropriate strain stimulation could induce BMSCsdifferentiate into TCs, and the best conditions of strain stimulation need more experiments.