In Vitro Culture And Label Of Fibroblasts From Rabbit Vocal Fold And The Study On Vocal Fold Scarring

Vocal fold scar that occurs following trauma or disease alter the biomechanical properties of vocal fold, stiffen the vocal fold, affect the viscoelasticity of vocal fold lamina propria(viscoelastic strength). All of the above change the biomechanical properties of the vocal fold, lead to decreased mucosal wave of vocal fold during vibration, therefore affect voice production. At present, optimal vocal fold scar prevention and treatment approach has not been found. In recent years, tissue engineering technology has made remarkable progress, the main principle is amplifying a small amount of seed cells in vitro and compounding with biological materials, cell factors to repair larger tissue or organ defects and to reconstruct the physiological function of tissues and organs. The method of tissue engineering technology would be used in laryngology for the regeneration of vocal fold, and even the regeneration of larynx in future. Human vocal fold is a highly differentiated layered structure. The histological structure of the vocal fold is divided into three layers:the epithelium, lamina propria and muscle layer (thyroarytenoid muscle). The lamina propria of vocal fold includes the superficial layer of the lamina propria(SLLP, Reinke's layer), the middle layer of the lamina propria(MLLP), and deep layer of the lamina propria(DLLP). The definition of the layers is significant in the description of the vocal fold structure and function. Lamina propria is the most important structure of vocal fold, composed of cells and extracellular matrix. Lamina propria is composed of a smaller number of cells, including fibroblasts, myofibroblasts,and macrophages. Fibroblasts are main cells to maintain the structure and function of lamina propria. These cells mainly locate in the DLLP. Their fuctions are to regulate degradation and re-distribution of cell interstitial sedimentary, extracellular matrix includes fiber protein and gap junction protein.In the first part of this study, we produced a rabbit model of vocal fold scar, and did a preliminary study on vocal fold scar. In the laryngoscope, the front and middle part of the bilateral vocal fold were clamped by the laryngeal forceps(fail to reach the muscle), no intervention was as control group, we sacrificed all the animals after 1 month, harvesting vocal folds, using Masson trichrome staining of to observe collagen, using Alcian blue staining to observe hyaluronic acid of the lamina propria. Using Image-Pro Plus 6.0 software to semi-quantitative analysed on the stained sections, the results showed that collagen of the vocal fold scar increased significantly, hyaluronic acid did not change significantly.In this study, we preliminary investigated the tissue engineering regeneration of lamina propria of the vocal fold. The three elements of tissue engineering is the seed cells, scaffolds, and cytokines. The second part of this research was the culture of seed cells-fibroblasts in rabbit vocal fold (vocal fold fibroblasts, VFFs) in vitro. We get the vocal fold of rabbit, used digestion method to obtain fibroblasts.The cells were cultured in high glucose DMEM containing fetal bovine serum medium in vitro, and we explored the best culture conditions. We observed the morphology and growth conditions of the cells under inverted phase contrast microscope, and the cells purified after passage. We detected fibroblast-specific protein Vimentin by immunohistochemistry, the positive rate of Vimentin was above 90%, indicating that the cells cultured are fibroblasts, and positive rate is high.The third part of this study we observed the compatibility and growth of allogenenic cultured cells. We labeled the cultured cells with Ad-EGFP.6 New Zealand rabbits, two rabbits were randomly selected as non-intervention group, the bilateral vocal fold of 4 rabbits were used as acute injury model, we injected concentration of 104/ml Ad-EGFP labeled cells into the left vocal fold, lml saline into the right vocal fold as control. After injection of the vocal fold, the rabbit were in good condition, no infection, laryngeal edema, immune rejection reaction. Vocal fold tissue were harvested after 15 days, fluorescence microscope found no survival VFFs. It is indicated that a simple VFFs'injection is difficult to survive in foreign tissue, we should provide scaffolds for them in order to be conducive to growth..In conclusion, we preliminary explored tissue engineering of the vocal fold,in this study, we finished the preparation of the seed cells, explored the compatibility of seed cells in vivo, and prepared the vocal scar animal model, in the follow-up study, we will build a cell-scaffold complex to implant into the vocal fold scar model, observe the regeneration and repair of vocal fold.