Experimental Study On The Small Intestinal Submucosa As A Scaffold For Tissue Engineering Tendon

【Objective】Porcine small-intestinal submucosa(SIS) scaffolds were processed by the physical and the chemical methods. SIS scaffolds were seeded with tendon cells (tenocytes) which were isolated from the embryonated egg. The constructs of the cell and SIS were implanted into the defect flexor tendon models of the adult chickens, we compared them with the only SIS to determine the differences between the two groups, and initially studied the feasibility of the small intestinal submucosa as a scaffold for tissue engineering tendon.【Methods】1. We got fresh jejunum from healthy porcine, after clearing, then cut the jejunum every 10cm. The tunica mucosa, serosa and tunica muscularis were abraded by longitudinal wiping motions with a scalpel handle wrapped with moistened gauze. SIS, processed by chemical methods, was curled as the axis, diameter of 1.5～2.0mm, 2.0cm long, the free end was sutured with the11/0 micro-lines. SIS was processed by freeze drying, vacuum packaging, andγ-ray radiation. 2. Digital tendon incided from 15 chick embryoes were digested by enzyme, and the harvested tenocytes were of proliferation and passage. 3. To compare the activity of the various generations of the cells. Additionally, we studied the proliferation of the cell in the first generation by MTT test. Growth curve was made. 4. The cells were seeded in the SIS for 5×10~6/ml. The adhesion of the cell was observed by the somatotype microscope. 5. Twenty four healthy adult Roman chickens were divided into two groups randomly, cell-seeded SIS group(experimental group) and only SIS group(control group). Left digit was operated in this experiment. Every group had 12 chickens, skin in the flexor aspect was dissected in the middle and distal segments of digitus medius, about 4cm long. The subcutaneous tissue was cut and exposed the tendon. The flexor tendon was distally dragged and fixed with little pin, the tendon was cut in the 5mm distal to the pin. The prepared SIS was used to suture with the proximal broken end of tendon for "8"shape, strengthening by two interrupted suture, after 2cm long tendon was cut, the other end of SIS was sutured with the tendon, and dressing. Two groups were isolated and freelance, left digit was fixed with gypsum for 2weeks. The chickens were sacrificed at 3, 6, or 9 weeks after surgery. Eight specimens were harvested at the every point. 7. Observation of specimen in general. 8. To inspect the specimens by the somatotype microscope. 9. To inspect the histologic pattern of the specimens. 10. Mechanical test of the specimens.【Results】1. Tenocytes were discovered by inverted microscope, Cells begun to adhere after 4 hours, culture flask was covered with cells until 72 hours. At this time, cells must be serial subcultivation. Proliferation was more rapid after serial subcultivation, appearances of cell were more of conformity, for long fusiform shape, however, only at 4th passages, cell activity obviously descended. 2. The results of MTT show that the proliferation of the tenocytes was more rapid forl, 2days. The proliferation of the tenocytes was in the platform stage for 3 days. The sum of the tenocytes had the tendency of descent for 6 days. 3. Discovered by the somatotype microscope, SIS extracellular matrix graft degraded rapidly, with approximately 60％of the mass lost by 3 weeks after surgery, and 90％of the mass lost by 6 weeks after surgery, the graft was completely absorbed by 9 weeks after surgery. Vacant position was substituted for collagen. 4. Discorvery by the histology, a lot of fibrous tissues can be seen in the cell-seeded SIS group. At 6, 9 weeks, the alinement of fibers forming in the cell-seeded SIS group was better than that in the without cell group. 5. At 3 weeks, the mean mechanical strength of constructs with cell was169.1±8.5 and without cells was169.8±9.1(P＞0.05), there was no difference in the two groups. At 6 weeks, the mean mechanical strength of constructs with cell was 197.3±9.1, compared with without cells 172.7±13.7(P＜0.05=, at 9 weeks, mechanical strength in the cell-seeded group was 243.5±7.8, compared with without cell-seeded group192.7±9.3 (P＜0.05=. Despite the mechanical integrity of these constructs was significantly less than native tendon, seeded groups were significantly more different than that of incubated only SIS in 6, 9 weeks.【Conclusion】1. The great reproductive capacity of cell was 2nd and3th generations. Cell appearance was of regularity. Cell had maximal secretary role. So we chose the cells of 2nd generations to seed in the SIS. 2. Aging was earlier than others. 3. Small intestinal submucosa extracellular matrix is rapidly degraded after implantation for the repair of a musculotendinous tissue in this tendon repair model and is rapidly replaced by the deposition and organization of host tissue. 4. Cell-seeded SIS scaffold was better than the without cell-seeded SIS. Cell-seeded SIS scaffold may be the suitable substitution for the defected tendons.