Experimental Study Of Flexor Tendon Allografts Cryopreserved By Vitrification

Objective To find out the appropriate conditions of vitrification to preserve flexor tendon allografts in chicken.And compared with freezing allografts in vitro and in vivo, including tendon cellular viability, content of hydroxyproline, immunogenicity and biomechanics. At the same time, to explore the feasiability of its clinical application.Methods 1) Screening and Detection: To use the orthogonal design and rapid detection technique of tendon viability, found out the appropriate conditions of vitrification and freezing to preserve flexor tendon allografts in chicken's toes. 2) Cell culture: Flexor tendons were preserved by vitrification and freezing, then tendon grafts were done under the cell culture, to observe its cellular viability by comparing with fresh tendon. 3) Detection in vitro: The morphologic changes, the content of hydroxyproline and mechanical capability of vitreous tendon grafts were observed or detected, which in order to comprehend what happened after cryopreservition. By means of comparing with fresh tendons and frozen tendons in vitro, thus we could know the benefit and shortcoming of vitrification method. 4) Detection in vivo: The vitreous flexor tendons were aliotransplanted in chicken's toes. After operation, the chicken were sacrificed in 2,4,8,12 and 16 weeks, and grafts were harvested to observe the morphological changes, immunologic rejection, content of hydroxyproline and biomechanical changes. By means of comparing with fresh flexor tendon autografts and frozen flexor tendon allografts, to evaluate the feasibility of vitrified tendons being used as allografts. 5) Long-term preservation: The half a year vitrifying tendon grafts were observed on the morphological changes, content of hydroxyproline and biomechanical capability in vitro, followed by bacteria culture of tendons and preservation solution. Then vitrified tendon grafts of half a year were aliotransplanted, and the changes of morphology, content of hydroxyproline andbiomechanical capability in 16 weeks were observed or detected. And to compare with the vitrified tendon allografts that were preserved for two weeks, to evaluate the long-term stability of vitrification.Results 1) Screening and Detection: Through methode which tendon was digested by enzyme, we got fresh tendon's viability was 89.26%, and tendon of vitrification maintained 78.49%, which was significantly better than 49.63% of freezing. 2) Cell culture: The cells that cultivated from vitrified tendons were tendon cells, which growing out from tissue in the 8th day, cells passaged in the 21st day. And counting of cells was 1.15×10~6. The cells that cultivated from frozen tendons were also tendon cells, which growing out from tissue in the 12th day, cells passaged in the 28th day. And counting of cells was 0.75×10~6, 3) Detection in vitro: The tendon cells and extracellular matrix could be preserved perfectly by vitrification, and which was better than freezing method. However the content of hydroxyproline among the fresh, vitrified and frozen tendons was no difference. And lag loop and stress relaxation phenomenon could be found among the three kind of tendon. And biomechanical capability among the fresh, vitrified and frozen tendons was no significantly different.4)Detection in vivo: The fresh, vitrified and frozen tendons were transplanted into chicken's toes by autografts and allografts, and wound got good healing, but there was middling level or even more heavy adhesion around tendon. Tendon cells in the vitrified tendon decreased significantly at 4 weeks after transplantion, and in the part of connection there were a great quantity small collagen fibers and fibroblast to fill in. Quantity of cells in the vitrified tendon had already approach to the normal at 8 weeks. And in the part of tendon connection, quantity of tendon cells significantly increased and small and thick collagen fibers arranged at interval at 16 weeks, and processes of reconstruction was still continued in tendon grafts.The content of hydroxyproline in the middle of tendon grafts decreased before 8 weeks, then gradually increased. However the content of hydroxyproline in the part of tendon connection gradually increased following with time,but there was no significantly different among the fresh, vitrified and frozen tendons. Three kind of tendons still belong to viscoelastic body,and lag loop and stress relaxation phenomenon were similar.Through the rupture test, we found the area of cross section in the part of tendon connection gradually decreased following with time. On the contrary,...