The Experimental Study On The Treatment Of Growth Plate Injuries With A Tissue-engineered Autologous Growth Plate

Bone bridge will form between epiphysis and metaphysis if the growth plate of long bone is injured by wound ,infection and tumor et al. The shortening and angulation of limbs caused by bone bridge is one of the big problems which many orthopedists encounters. In 1967,Langenskiold first reported that the partly early closure of the growth plate can be treated by the method of bone bridge cutting and fat packing .Since that ,people attempted to solve the problem by transplanting different grafts such as silica gel ,bone wax , fat and free growth plate et al. The above methods have some effects ,but they also have many defects. Immune expelling between the supply and the recipient is one of the dilemmas. Fat, muscle and bone wax only can prevent bone bridge forming caused by gentle injury instead of the growth plate regenerating, they also can not improve the growth and development of the limbs. In recent years, people try to solve the problem by the method of tissue engineering. We attempt to treat the injury of long bone growth plate by using the tissue-engineered autologous growth plate established in vitro. The experiment aims to establish the foundation for the clinical solving of the tough problem. 1.The culture of the iliac growth-plate chondrocytes andthe study on its cartilage-formation role in vivo Objective:To establish a kind of iliac growth-plate chondrocyte culture and storage method so that we can obtain the seed cells in large quantity for the usage of engineered growth plate, tissue.To observe the cartilage formation of the cells in vivo. MethodsrThe cells were harvested from the iliac growth-plate cartilage of the two-week old rabbit and cultured. Their biological phenomenons were observed and their biological characteristics were identified by using toluidine blue, alkaline phosphatase staining and immunohistochemistry staining for type II collagen. The second generation cells were harvested and injected into the dorsum of nude mice. Specimens were harvested 4 weeks later for histological examination. Results:The cells could proliferate in vitro and have no apparent de-differentiation within the sixth generation, but they could not differentiate into the hypertrophic cells. The cells revived from being frozen(-196癈) was 92%. The cells injected into the body of the nude mice could form the cartilage-like tissue. The iliac growth-plate chondrocytes can differentiate into hypertrophic cells in vivo. Conclusion:We have established a method of culture and storage of iliac growth-plate chondrocytes and cartilage-formation of the cells in vivo was verified. The cells can differentiate into hypertrophic chondrocytes in vivo.2.Experimental study on the establishment of tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cellsObjective:To investigate the feasibility of establishing tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells. Methods: We obtain the growth plate cells from iliac crest epiphyseal cartilage of the three-week old New Zealand rabbits by dissection and sequential digestion with 0.2% collagenase(type II).After the cells proliferated with monolayer culture in vitro for three weeks.The cells were harvested and cocultured with the scaffold DBM.The cell-material complex was observed by "HE" staining and electronic scanning microscope in order to evaluate the interaction between cells and DBM. Results: Twenty-four hours after coculture, the cells adhered to DBM scaffolds; Seven days later, the growth-plate chondrocytes proliferated in the DBM network and secret the extracellular matrix; The cells grow all formed on the surface of the scaffold after three weeks. Conclusion: We can successfully establish the tissue-engineering growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells .3.The experimental study on the treatment of growth plate injuries with a tissue-engineered autologous growth plate Objective.