Reconstruction Of Cartilage Tissue With Biological Substitutes Using Tissue-engineering Technique

In modern head and neck surgery, there is growing demand for a material that can be used to replace large cartilage defects. Traumatic, tumorous, or congenital lesions of the midfacial region or rhinobasis need to be reconstructed carefully to guarantee sufficient aesthetic and functional results. Furthermore, the replacement of larynx and trachea still remains an unsolved problem. Until now, no material have been found that could fulfill all the demands or the main ones of a tracheal equivalent.Today, autologous rib and ear cartilage is widely used due to the remaining transplant viability and only minor resorption phenomena. Unfortunately, there is rarely enough material. In addition, the risks of a second operation have to be considered. Furthermore, calcification mainly of rib cartilage would have a negative influence on mechanical characteristics.Allogeneic transplants must be conserved to prevent autolysis. At the same time, antigenic characteristics and the chance of transmitting infection are eliminated or at least reduced. However, no definite consensus exists regarding whether transmission of infective chemicals. Xenogeneic transplants need to be treated for conservation as well, which results in frequent resorption, moreover, the risk of infectiousness cannot be excluded completely. The poor healing potential of cartilage tissue has been ascribed in part to a paucity of chondrogenic cells available for repair.Tissue engineering is an interdisciplinary field that applies the principles of engineering and the life sciences toward the development ofbiological substitutes that restore, maintain, or improve tissue function. Articular hyaline cartilage ^ human septal cartilage and cartilage in the shape of a human ear had been engineered in vivo by tissue-engineering technique. This study evaluated the feasibility of growing tissue-engineered cartilage in the shape of a human trachea and sheet. The preliminary results were as follows:In the first part of the study, we constructed three dimentional biodegradable polymer foams of PLGA using laminating technique, then tested the physical characteristics of PLGA foams. The test value listed as follows: pore diameter 180-350urn, porosity 85%. Then, the testes of cytotoxicity and intramuscular implantation were carried out to evaluated biological effects of polymer foams of PLGA. The primary results showed that ploymer foams of PLGA had good biocompatibility in vitro, which were no significant toxicity to body. These results exhibit that the novel porous PLGA foams may be suitable for use as a bioerodible scaffold for regeneration of cartilage tissue.In the second part of the study, freshly slaughtered rabbit forelimbs were obtained, and cartilage fragments were sharply cut off the articular surface of each joint. Using a method similar to that described by Klagsbrun, the fragments were subjected to collagenase digestion (2mg/ml), and the acquired cells were cultured in DMEM containing 10% fetal bovine serum. Cells grew to confluency in about 5 days, and then were subcultured. The cell type was identified as chondrocyte by the cell morphology and the specific traits of chondrocyte. The stability of the chondrocyte growth could last for about 8 passages. From passage 3 to 8, the chondrocyte showed a stable proliferation with 36 hour of the doubling time. So the method is ideal for the primary culture of chondrocyte. Whencell number and viability were determined, chondrocyte suspensions were concentrated to a cellular density of 2x107 cell/ml, and were then seeded onto PLGA foams to form chondrocyte-polymer constructs. The constructs were either cultured in RCCS system, or in plate. Specimens were examined grossly and then for histologic analysis, the results showed that the constructs cultured in RCCS system exhibited significant cartilage formation. In contrast, the specimens in plate showed little cartilage formation only. We found that RCCS culture system made cells grown under the most natural conditions possible.In the third part of the study, the PLGA foams produced by laminating technique were shaped into the form of a human trachea or sheet. Then chondrocyte were isolated and cultured, chondrocyte suspentions were concentrated to a cellular density of 2x107 cells/ml. When the PLGA foams were shaped, chondrocyte were seeded onto each polymer matrix in experimental groups I and II, and allowed 4 hours to attach to the polymer, the cell-polymer constructs were then incubated in RCCS system or plate for 1 week. Under general anesthesia and employing sterile surgical technique, each constructs was implanted into subcutaneous pockets of athymic mice. The animals were sacrificed and speciments were dissected free of surrounding soft tissue for gross and histological analysis. The results showed that when cultured in RCCS system, the constructs formed cartilage in the shape of a human trachea or sheet in vivo, but the constructs in plate showed little cartilage formation in vivo only, this results demonstrated that the RCCS system can offer the most natural conditions possible for the formation of cartilage.