Experimental Study On The Repair Effect Of Autologous Platelet-rich Plasma On Condylar Cartilage Injury In Growing Rabbits

Objective:During the growth and development stage,many factors may cause condylar cartilage damage,which will bring with a serious negative impact on the growth and development of condyle and mandible.Further more,it not only brings a lot of inconvenience to patients' lives,but also brings them great psychological burden.Therefore,it is very important to repair the condylar cartilage injury in time.However,there are many deficiencies in the treatment of condylar cartilage injury in growing stage.So,the purpose of this study was to observe the histological and molecular changes of autologous platelet-rich plasma in repairing the condylar cartilage injury in growing rabbits at different times and to explore the repairing effect of platelet-rich plasma on the condylar cartilage injury in growing stage.So as to provide theoretical basis and treatment plan for the treatment of condylar cartilage injury in growing and developing stage.Methods:1.Establishment of the animal model:An animal model of condylar cartilage injury was established in New Zealand white rabbits.Under general anesthesia,a circular cartilage defect area with a diameter of 3 mm and a depth of 2 mm was prepared at the center of the mandibular condylar joint with the dental fissure bur.2.Experiment grouping:The rabbits were randomly and averagely divided into three groups as fellowing:sham operation group(no cartilage defect),injury group(received normal saline)and PRP group(received PRP treatment).The animals were sacrificed at 6 and 12 weeks through excessive anesthetic injection.3.Preparation and activation of autologous PRP:PRP was prepared through twice centrifugation using 10 m L fresh whole blood.First step centrifugation at 250*g for 10 minutes and retain all supernatants and 1.0-2.0 mm below the interface for second step centrifugation at 1000*g for 10 minutes.After the centrifugation,PRP gel was prepared through activation.4.Platelet counting:The whole blood and PPR were stored in ice box and sent to the Laboratory Department of Stomatology Hospital Affiliated to China Medical University for platelet count.The platelet content in different samples analyzed by automatic blood cell counter to verify whether the platelet quantity in the PRP prepared by this method met the experimental requirements.5.Observation results and evaluation indexes:At 6 and 12 weeks,six rabbits in each group were executed by injecting excessive anesthetics.The International Cartilage Repair Society(ICRS)macroscopic scoring were used to assess the general score.Histological examination was performed by hematoxylin-eosin(HE)and safranine-fixing green staining,and ICRS histological score was used to evaluate the results.Western blot and q-PCR were used for molecular detection.Results:1.Platelet counting:The number of platelets in PRP is(5.55±0.16)times that in whole blood,which meets the requirement of PRP preparation by secondary centrifugation and meets the current experimental requirements.2.General situation and observation:At 6 weeks,the defect area in PRP group was filled with neoformative tissues.The condyle developed well and there was no significant change in size and shape.At 12 weeks,the cartilage defect area was basically repaired in PRP group.Since the fusion of the neoformative chondroid tissues and the surrounding tissues,there were no obvious signs of cartilage damage.Further more,no obvious hypodevelopment of condylar and mandible was observed as well.ICRS score showed that the degree of cartilage repair in PRP group was significantly better than that in injury group at 4 and 8 weeks after operation(P?0.05).3.HE staining:At 6 weeks,the defect area in injury group was covered by few arranged fibrous tissue with rough surface while the defect area in PRP group was well filled with neoformative tissues,where the surface was reasonable flat and the height was basically the same as the surrounding tissues.A few fissures were found at the junction between the neoformative tissues and the surrounding tissues.At 12 weeks,the fibrous tissue increased significantly in the injured group while the defect area in PRP group was favorable repaired and fused well with surrounding tissues.4.Saffron O-Fixed Green staining:At 6 weeks,no saffron O staining was found in the defect area in the injury group while there was a weak saffron O staining at the bottom of the defect area in PRP group,which was thinner than the surrounding normal tissues.At 12 weeks,a small amount of extrachondral matrix was observed in the defect area in the injury group while obvious saffron O staining was observed at the bottom of the defect area in PRP group,which was similar to the surrounding tissues.However the content of hyaline cartilage in PRP group was still less than that of normal tissues.5.Western blot:The expression levels of Col I,Col II,Sox9 and Agrecan protein in PRP group were higher than those in injury group at 6 and 12 weeks.6.q-PCR:At 12 weeks after operation,the relative expressions of Col I,Col II,Sox9 and Agrecan in PRP group were significantly higher than those in injury group(P?0.05).Conclusion:It is difficult to repair the injured condylar cartilage in growing period,but activated autologous PRP can effectively repair condylar cartilage injury in growing period.Autologous PRP may be an ideal material for repairing condylar cartilage injury in growing period.