| ObjectiveTo esteblish experimental osteochondral tissue bank abided by AATB standards with slow-cooling gradient cryopreservation, observe cell viability and cell metabolism of preserved cartilage, and clinical application of osteochondral allgraft for three cases.MethodsThe human articular cartilage specimens came from the clinical brain death patients of Qianfoshan hospital,Shangdong province.The age of donors was controlled at 15-45 years old.Donors, blood and urine were checked before death within 12 hours under aseptic conditions. Arthrex device of osteochondral transplantation was used to dill to cut for 200 pieces of osteochondral plugs from both femoral condyles and tibial plateau by the surgical procesure. The specimen were steriled to package individually after processed by the 10%DMSO,then used slow-cooling gradient to - 80℃and preserved in - 80℃refrigerator,so osteochondral tissue bank was established. 10 specimens were took out randomly at the time of 1 month, 3 months, 6 months and 1 year,and detected chondrocyte survival rate by placenta blue staining, MTT assay for cartilage cell activity; 10 specimens with histological and histochemical to observe chondrocyte morphology and metabolic activity. The fresh cartilage as control, conducts the comparison research to each group viability and metabolism of chondrocytes;take 6 samples to treat knee cartilage defects for three knee cartilage defect cases, and evaluate the clincal results.Results1.Changes of chondrocyte survival rate: The fresh group was 94.80±4.59,the viability of cartilage cryopreservation group of at the time of one month,three month,six month and one year were 66.95±9.98,58.11±9.11,43.74±8.37,29.21±5.03, compared with the fresh group each group had significant statistical difference(P<0.05),and pairwise comparison of the experimental groups at different check time had significant statistical difference(P<0.05).The viability of chondrocyte wds depend on the time of cryopreservation.2.Changes of articular cartilage PG: The PG content of fresh group was plentiful, Compared with the control group, the PG content of grading-cooling cryopreservation group at the time of one month,three month,six month and one year had significant statistical difference(P<0.05),and pairwise comparison of the four groups at differrent time had significant statistical difference(P<0.05). PG content was depend on the time of cryopreservation.3.Activity of cartilage: OD value of the fresh group was 0.65±0.03,the cryopreservation group at the timeof one month,three month,six month and one year were 0.42±0.04,0.29±0.02,0.21±0.03,0.16±0.04,compared with the control group,the PG content of grading-cooling cryopreservation group at different time had significant statistical difference(P<0.05), comparison of the four groups in terms had significant statistical difference(P<0.05). Cryopreservation effected on activity of cartilage over time.4.The osteochondral specimens taken under aseptic conditions had been preserved for one month to one year,the pakage were intact, the bacterial culture test results of selected specimens randomly were negative; 6 specimens had been used for articular cartilage defects of knee,and the postoperative MRI shows that the grafts and host bone fusion was well,the Brittberg-Peterson function score was significantly lower than preoperative, shows clinical satisfactory in primary.Conclusions1.The viability of cryopreserved articular cartilage had dynamically time compliance,the metabolism of articular cartilage wasdecreasing with time gone;frozen lead to cartilage frostbite was objective, the slow-cooling gradient cryopreservation could protect the cell viability maxmium.2.Grading-cooling cryopreservation was operable,and could extend cell survival of allgraft, the cartilage tissue bank creation was feasible. 3.The application of cryopreservation allagraft for articular cartilage defects surgical treatment achieved initial results, and provide the basis for clinical application. |
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