Preliminary Experimental Study On Immune Rejection Of Particulated Juvenile Allograft Cartilage Transplantation

OBJECTIVE: To establish a large animal model of full-thickness cartilage defect of the joint using Guizhou minipig as experimental animal,compare and analyze whether there is a difference between the immune rejection response induced by particulated juvenile allograft cartilage and the inflammatory response induced by autologous cartilage chips transplantation,and to provide experimental basis for clinical application of particulated juvenile allograft cartilage.METHODS: Twenty-four adult Guizhou pigs were selected as experimental animals and randomly divided into experimental group and control group,with 12 in each group.The adult minpigs used in each group were numbered by random numbers.The odd number selected the right knee for surgery,and the even number selected the left knee for surgery.Four young Guizhou minipigs(<2 months old)were selected as the source of particulated juvenile allograft cartilage in the experimental group.The experimental group used particulated juvenile allograft cartilage,and the control group used autologous cartilage chips.The experiment was performed in 4 batches,each batch of the experimental group and the control group used 3 adult minipigs,an additional juvenile pig was used in experimental group per batch.In the experimental group,each juvenile piglet was anesthetized by intravenous injection of sodium pentobarbital via ear vein and sacrificed.The knee joint capsule was dissected under aseptic conditions,and the knee cartilage was peeled from the articular surface to prepare about 1 mm3 cartilage microparticles,weigh 3 parts of cartilage microparticles,1g each,and put them into 3 centrifuge tubes with sterile saline solution for storage,and then implanted them into cartilage defects one by one within 2 hours that were freshly prepared in 3 adult pigs on the same day.The adult minipig was induced muscle relaxation by injecting xylazine intramuscularly and anaesthetized by injecting pentobarbital sodium via ear vein.The operation position was lateral position,with the operation limbs of minipig above.Shave the operation area after satisfactory anesthesia,sterilize the knee area with iodophor,lay a sterile surgical towel,and fix it with forceps.Under sterile conditions,use a scalpel to cut the skin of the knee joint in the side limb,cut in layers,and bluntly separate the knee capsule.Slit the knee capsule until seeing the patella.Pull the patella to expose the femur trochlea,use an 8mm diameter circular saw perpendicular to the articular surface to prepare a full-layer articular cartilage defect model,optimize the defect with a curette and peel off the articular cartilage,rinse the cartilage defect with sterile saline to remove residual cartilage debris.The juvenile cartilage microparticles prepared in advance on the same day were mixed with the fiber gel and implanted into the cartilage defect prepared from the allogeneic group.In the control group,the articular cartilage defect was prepared using the same surgical method as the experimental group,and the cartilage collected from the knee joint was cut into a cartilage particle of about 1 mm3 with a scalpel and scissors,and 1 g of cartilage particles was weighed.Put it into a centrifuge tube filled with sterile saline,wash it,mix it with the fiber gel,and then replant it in situ in the articular cartilage defect prepared by the autograft group.After surgery,the joint capsule,fascia,and skin tissue were sutured layer by layer.Each piglet received intramuscular injection of ceftiofur sodium(0.5g)to prevent infection,twice a day for one week.Six adult pigs were sacrificed in each group at 1 and 3 months postoperatively.The knee joint fluid was collected,and synovial and osteochondral samples were collected for analysis.These samples were fixed with 4% paraformaldehyde,and the osteochondral samples were decalcified with EDTA solution.All of them were embedded in paraffin,sectioned,and HE stained and immunohistochemically stained.Observation indicators include: gross score of cartilage defect area,analysis of synovial fluid cells,HE staining to observe inflammatory cell infiltration,and immunohistochemical staining to analyze the expression of TNF-? and IL-6 in synovium and articular cartilage.RESULTS: No abnormalities such as wound infection,wound non-healing,and sudden death were observed.In the first month after operation: the articular cartilage repair score of the control group was slightly better than that of the experimental group,but the difference between the two groups was not statistically significant.The results of HE staining of articular cartilage and joint synovial membrane showed that the number of inflammatory cells in the experimental group was more than that in the control group,but the difference was not statistically significant;the results of IL-6 immunohistochemistry showed that the expression in the experimental group was more than that in the control group,but the difference was not statistically significant;The expression of TNF-? in the experimental group was statistically higher than that in the control group(P < 0.05).In the third month after operation,the general score of cartilage repair in the two groups was higher than that in the first month,there was no significant difference between the two groups.The number of synovial inflammatory cells,cartilage and synovial inflammatory cells,and the expression levels of TNF-? and IL-6 in the experimental and control groups were all lower than that in the first month.The difference between the two groups narrowed,and the difference was not statistically significant.Conclusion: A full-thickness articular cartilage defect model was successfully established using Guizhou minipigs.The use of particulated juvenile allograft cartilage transplantation and autologous cartilage chips transplantation to treat full-layer cartilage defects both evoked local reactions,there was no significant difference between the immune rejection response induced by particulated juvenile allograft cartilage and the inflammatory response induced by autologous cartilage chips at 3 months after surgery,providing a large animal experimental basis for the clinical application of this technology.