Experimental Study On Biological Change Of Different Forms Of Autogenous Costal Cartilage After Transplantation

Objective:In recent years,costal cartilage has been widely used in clinic because of its unique advantages.This experiment mainly compared the changes of different forms of human costal cartilage after autogenous free grafting by histological and molecular biology research methods,which plays a certain guiding role in the selecting different forms costal cartilage in clinic.Methods:According to the inclusion and exclusion criteria,8 patients with congenital microtia were included,including 6 males and 2 females,with an average age of 15.7 years（15.7±1.7）.The remaining autologous cartilage after fabrication of auricle framework in the first stage of auricle reconstruction was collected,made into different forms and then subcutaneously transplanted into the chest wall.The transplanted autologous cartilage specimens were collected in the second stage of auricle reconstruction.Grouping:autologous cartilage before free transplantation was used as control group（group A）,while autologous cartilage after free transplantation for 6 months was used as experimental group.The experimental group was divided into two groups:Ba and Bb.Based on the thickness of transplanted autologous cartilage,Ba group was subdivided into three groups:Ba₁,Ba₂and Ba₃,and the thickness of which was 2mm,4mm and 6mm,respectively.Group of Bb was divided into block（Bb₁group）and granular（Bb₂group）according to the shapes.The autologous costal cartilage grafts were initially observed by macroscopic observation of whether there was a general change after transplantation.Further,the activity and morphology of chondrocytes,the number of chondrocytes,the distribution of glycosaminoglycans and type II collagen fibers in the cartilage matrix of different groups were observed by HE staining,Safranin O-fast green staining and Sirius scarlet staining under microscope.The morphology of cartilage lacunae,the integrity of chondrocytes and the arrangement of cartilage matrix in different groups were further observed by SEM.Finally,the expression of GAG chain synthesis related gene mRNA and collagenⅡmRNA was detected by RT-qPCR and the expression of collagenⅡprotein was detected by WB in cartilage matrix.Statistical data were analyzed to compare whether there were differences in the histology and molecular biology of autologous costal cartilage in group A,Ba and Bb.Results:（1）The results of macroscopic observation:there was no significant change in Ba₁group,Ba₂group,Ba₃group and Bb₁group.Most of the autologous costal cartilage grafts had no significant change in Bb₂group,and a few of them had blunt edges.（2）histological results:（1）There was significant difference in the number of chondrocytes among A group,Ba₁group,Ba₂group and Ba₃group（F=8.377,P<0.001）.Compared with group A,the number of chondrocytes in Ba₁group and Ba₂group were significantly lower than that in group A（P<0.01）,but not in Ba₃group（P>0.05）.There was no significant decrease in chondrocyte count in Ba₁group,Ba₂group and Ba₃group（P>0.05）.（2）There was significant difference in the number of chondrocytes among group A,Bb₁and Bb₂（F=17.174,P<0.001）.Compared with group A,the number of chondrocytes in Bb₁group and Bb₂group decreased significantly（P<0.001）.The number of chondrocytes in Bb₂group was significantly lower than that in Bb₁group（P<0.05）.（3）RT-qPCR detection results:（1）There was no significant difference in Galnac4s6st mRNA expression among group A,Ba₁,Ba₂and Ba₃（F=0.688,P=0.566）.（2）There was significant difference in Galnac4s6st mRNA expression among group A,Bb₁and Bb₂（F=21.964,P<0.001）.Compared with group A,the expression of Galnac4s6st mRNA increased in Bb₁group（P=0.028）,but decreased in Bb₂group（P=0.004）.The expression of Galnac4s6st mRNA in Bb₂group was lower than that in Bb₁group（P<0.001）.（4）There was no significant difference in CollagenⅡmRNA expression among group A,Ba₁,Ba₂and Ba₃（F=1.243,P=0.303）.（4）There was no significant difference in CollagenⅡmRNA expression among group A,Bb₁and Bb₂（F=2.927,P=0.065）.（4）WB detection results:（1）There was no significant difference in the average relative expression of CollagenⅡprotein among group A,Ba₁,Ba₂and Ba₃（F=1.615,P=0.212）.（2）There was no significant difference in the average relative expression of CollagenⅡprotein among group A,Bb₁and Bb₂（F=0.283,P=0.757）.Conclusion:After 6 months of autologous costal cartilage transplantation,the number of chondrocytes increased with cartilage thickness and size,and GAG content increased with cartilage size.The thickness and shape of the cartilage did have an effect on the microstructure of the cartilage after transplantation,but had little effect on the expression of mRNA and protein in the cartilage matrix.In clinical application,costal cartilage can be made into various shapes for transplantation.