The Study On Preservation Methods Of Human Osteochondral Allograft

PurposeThis issue compares different effects of six different preservation methods on morphology of articular cartilage, the survival rate and activity of the cartilage cells. These six different methods which making fresh human articular cartilage as a control group are gradient cooling cryopreservation, constant cooling cryopreservation, vitrification, direct liquid nitrogen, Co⁶⁰ irradiation and gradient cooling cryopreservation and alcohol immersion preservation method. In terms of the comparative study, select the best preservation method, providing a cartilage bone graft for the clinical application.MethodsThe human articular cartilage specimens came from the clinical brain death patients of affiliated hospital of Taishan medical college and Qianfoshan'hospital .The ages of patients were controlled at 15 to 45 years old. Patient'blood and urine were checked before death. Under the aseptic conditions, use hollow drill to cut for 500 pieces of osteochondral plugs from both condyles of femur and tibial plateau. Their diameter was 4.5mm and 5mm of thickness, including some subchondral bone.The osteochondral plugs were washed 3 times with PBS. The specimen would be randomly divided into 7 groups, including the control group, gradient cooling group, Co⁶⁰-irradiation group and gradient cooling, the vitrification group, constant cooling group, the direct liquid nitrogen group, alcohol immersion preservation group. The control group did not take any measures. Directly detect the survival rate and the activity of cartilage cells and observe the ultrastructure in 20min. Gradient cooling group was a method which put specimens to 4 temperature gradients before it droped to -40℃. Co⁶⁰-irradiation group was to irradiate osteochondral plugs with Co⁶⁰-ray for two hours, afterward preserve it with the gradient cooling method. The vitrification group was that the specimen was immersed by the vitrification solution and then put it into liquid nitrogen at the fastest speed to preserve. Continuous cooling group was the traditional cooling method, that is the specimen immersed by the low-temperature protective agent was preserved in two-steps. Direct liquid nitrogen group was to put osteochondral plugs immersed by low temperature protective agent directly into liquid nitrogen to preserve. Alcohol immersion group was about to soak the plugs into 75% alcohol for 72-hour, and then replace by 95% alcohol under 4℃to preserve. Cartilage specimens in all experimental groups took out in the preservation of 8 days, 15 days, 30 days,60 days and then used trypan blue to detect chondrocyte survival rate and use MTT to detect activity of cartilage cells (OD values reflect the activity of cartilage cells); with histology and histochemical detection, observe morphological changes and metabolic activity of cartilage cells (Safranine O staining showed that cartilage matrix PG Change); observe the changes of the ultrastructure with TEM. And make the fresh group as the control group to compare with the experimental group.Results1,Changes of articular cartilage PG: Preservation of articular cartilage for 8 days, compared the PG content (IOD) values of the cooling gradient group, the Co⁶⁰-irradiation group and the constant cooling group with the control group, there was significant statistical difference (P <0.05), and there was no statistical difference among the experimental groups. Preservation of articular cartilage for 15 days, compared with the control group, the PG content of slow cooling gradient group, Co⁶⁰-irradiation group and constant cooling group had significant statistical difference (P <0.05), and there was statistical difference between the cooling gradient group and the Co⁶⁰-irradiation group, the constant cooling group. Preservation of articular cartilage for 30 days, there was significant statistical difference between the cooling gradient group and the Co⁶⁰-irradiation group, the constant cooling group (P <0.05); Preservation of articular cartilage for 60 days, made pairwise comparison of the cooling gradient group, the Co⁶⁰-irradiation group and the constant cooling group, there was statistical difference. the cooling gradient group, the Co⁶⁰-irradiation group and the constant cooling group, at the four time points, the three experimental groups compared in terms, the influence of preservation treatment to PG had time compliance, preserving in 8 days, 15 days, 30 days and 60 days, pairwise comparison of the three groups in terms had significant statistical difference (P <0.05). The PG content of vitrification group, direct liquid nitrogen group and the alcohol immersion group had no statistical significance. Compared the direct liquid nitrogen group with the control group, there was statistical difference. The vitrification group and the alcohol immersion group had no statistical difference at different time points.2,Changes of chondrocyte survival rate: The chondrocytes survival rate of control group was 96.63±0.56, preserved 8 days, 15 days, 30 days and 60 days, compared the experimental groups with the control group, there was statistical difference (P <0.05), pairwise comparison among the experimental groups had significant difference (P <0.05). Except alcohol immersion group, pairwise comparison of the experimental groups in terms had significant statistical difference (P <0.05).There wasn't any active chondrocyte in alcohol immersion group.3,Activity of cartilage cells: OD value of the control group was 0.66±0.05. Preserved for 8 days, the experimental groups compared with the control group had statistical difference (P <0.05). Compared slow gradient cooling group with vitrification group, Co⁶⁰ irradiation and gradient cooling group with slow constant cooling group, neither of them had statistical difference. The two former groups compared with the latter two groups had statistical difference (P <0.05). Preserved for 15 days, the experimental group compared with the control group had significant difference (P <0.05),compared Co⁶⁰ irradiation and gradient cooling group with the slow constant cooling group, there was not statistical difference, pairwise comparison among other groups had statistical difference (P <0.05);Preservation for 30 days was the same as the preservation for 15 days. Preserved for 60 days, compared the experimental groups with the control group, there was statistical difference (P <0.05), pairwise comparison among the experimental groups had significant difference (P <0.05). pairwise comparison of the three groups in terms had significant statistical difference, that was, the influence of preservation treatment to OD value of every experimental group had dynamic time compliance.Conclusion1 The methods of gradient cooling cryopereservation and vitrification which had some certain clinical application value could reduce the tissue frostbite of the articular cartilage significantly and improve the activity of preserved cartilages.2 Alcohol immersion method could only preserve the tissue structure of cartilage. Other preservation methods'effect on articular cartilage activity had dynamic time compliance, the activity of aricular cartilage was decreasing with time gone.3 Compared with gradient cooling method, Co⁶⁰-ray irradiation and gradient cooling method had damage on cartilage in a certain degree.