Ossification Mechanism Of Bone Bridge Formation After Physis Injury And Prevention Of Growth Arrest By Different Interpositions Into Physeal Injury

Section 1:The purpose of this investigation was to understand the molecular mechanisms of bony bridge formation with a proximal tibial transphyseal injury rat model. This study was to evaluate the in vivo cellular and molecular changes in the sub-injury cartilage adjacent to the transphyseal injury site on rat proximal tibia. Consisted with the histological changes, both Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and in situ hybridization experiment using Col2a1 probe showed there was the sub-injury cartilage region adjacent to the original injury site. Despite the sub-injury region remained normal cartilage structure and Collagen type X in the extracellular matrix, the chondrocytes within this region showed the dislocation with the cartilage lacunas, and the strand breaks of cleaved DNA in TUNEL assay. That these chondrocytes didn't express Col2a1 mRNA further confirmed they were dead cells. Along with the degradation of sub-injury cartilage, some fibroblast-like cells presented to the cartilaginous region between the sub-injury region and uninjured cartilage. In situ hybridization experiment for Patched 1 (Ptch1), indicator of Indian Hedgehog (IHH) signaling, indicated these fibroblast-like cells could respond to Hh signaling. These results suggest that the bony bridge formation involves series of changes of chondrocytes and Ihh signaling may be involved in the formation of the transient perichondrium-like structure between the sub-injury cartilage and normal cartilage, and partially contribute to the bony bridge formation.Section 2:To understand repair effects of fat and fibrin graft interposition with a proximal tibia transphyseal injury rat model, and to assess the reliability of treatment to physeal injury with the fibrin. A unilateral growth plate injury was created in the right proximal tibia of 28 rats without any graft interposition and the left was undealt. In other 28 rats, a bilateral physeal injury was made with the defect of the left tibia filled with autogenously adipose tissue and the right tibia filled with fibrin. To compare the malformed extents of different interventions, the length and the metaphyseal-diaphyseal angle of the tibia of different groups were examined. Further micro-CT assessment and histological staining were used to compare the bony bridge formation under different interventions. The deformity angle and medial length of the tibia were significantly different between grafted tibias and non-grafted tibias at 4, 16 and 24 weeks postoperatively (p<0.01). There was no significant difference between fibrin-graft and fat-graft tibias (p>0.05). Furthermore, the BMD of bony bridge induced by injured were significantly different between grafted tibias and non-grafted tibias at 4, 16 and 24 weeks postoperatively (p<0.01). Histological findings showed that physeal defects of the non-grafted animals were healed by bony bridge formation; however, the bony repair was inhibited by both fibrin and fat interventions. Fibrin could function as a substitute of adipose tissue in preventing the deformity induced by epiphyseal injury. Moreover, the utilization of fibrin as a scaffold will help us to develop some very effective intervention grafts to prevent skeletal deformity and diseases in future.Section 3:The aim is to understand the actions of VEGF in rat model after its proximal tibia transphyseal injury, and to evaluate the presumption of treatment to physeal injury with anti-VEGF-Flt. The growth plate injury model was created in 52 skeletally immature rats. Consisted with the histological changes, and in situ hybridization experiment using VEGF-Flt probe were used to study the bony bridge formation, expressing of VEGF of chondrocytes. The length and metaphyseal-diaphyseal angle of the grafted tibia were compared. Micro-CT assessment and histological staining were used to compare the bony bridge formation under different interventions. At injury site, VEGF signaling appeared on day 10 and increased as time went by. The deformity angle and medial length of the tibia were significantly different between anti-VEGF-Flt grafted tibias and non-grafted tibias at 2 and 4 weeks postoperatively (p<0.01). There were not significantly different between grafted tibias and normal group at 2 and 4 weeks (p>0.05), but significantly different at 16 and 24 weeks postoperatively (p<0.05). VEGF signaling may be involved in the formation of the bony bridge. Physeal bar may be inhibited by the use of anti-VEGF-Flt in forepart, but the inhibiting effects decrease as time goes by.Section 4: To understand repair effects of anti-VEGF combined with fibrin graft interposition with a proximal tibia transphyseal injury murine model, and to assess the reliability of treatment to physeal injury with the compound. The growth plate injury model was created in 60 skeletally immature rats. Consisted with the histological changes, and in situ hybridization experiment using VEGF-Flt probe were used to study the bony bridge formation, expressing of VEGF and the changes of chondrocytes. The length and metaphyseal-diaphyseal angle of the grafted tibia were compared. Micro-CT assessment and histological staining were used to compare the bony bridge formation under different interventions. At injury site, VEGF signaling appeared on day 10 and increased as time went by. VEGF signaling may be involved in the formation of the bony bridge. The deformity angle and medial length of the tibia were significantly different between grafted tibias and non-grafted tibias at 4, 16 and 24 weeks postoperatively (p<0.01). There was no significant difference between compound-graft and fat-graft tibias (p>0.05). There was no significant difference of the BMD between anti-VEGF-Flt grafted group or FS combined with anti-VEGF-Flt grafted group and fibrin-graft or fat-graft tibias (p>0.05). The long-term prevention effects of compound-graft was significantly better than that of others including fibrin-graft and anti-VEGF-Flt graft group, and little better than that of fat-graft group. Fibrin and anti-VEGF-Flt compound graft interposition would play a role in reducing growth disturbance in growth plate injury.