The Pilot Study Of Tissue Engineering Of Human Parotid Gland Constructed By The Composite Of Collagen And Chitosan

Parotid gland, submandibular gland, and sublingual gland are three primary salivary glands in oral cavity, could lubricate the mucosa of oral cavity and digestive tract, increase the taste sensation of mouth, and have various digestive enzymes to digest food, minerals to remineralization of teeth in saliva as well.So do some minor salivary glands such as labial gland, buccal gland , palatine gland. Salivary gland atrophy and secretion function disorder may cause the symptoms of dryness of mouth, secondary caries, long-term chronic mucosa inflammation,dysphagy and malnutrition, which may do harm to the patient's living quality seriously. About 500 thousand people worldwide were troubled by malignant head and neck tumors, experienced inadvertent damage and functional impairment of the salivary glands, which is due to the radiation therapy of malignant head and neck tumors while the salivary glands were in the radiation fields. In addition, degeneration of salivary glands with ageing, chronic inflammation, constantly application drugs which inhibit secretion function of salivary glands could lead to secretion function disorders of salivary glands that were difficult to recovery. Therefore, construction of artificial salivary gland to replace injured gland by tissue engineering theories and methods has become the field that researchers pay close attention to. In this study, we used HSG cells as the seed cellsmodel to explore whether degradating scaffold materials were suitable to construction of tissue engineering salivary glands and had good compatibility with seed cells, and to clear proliferation, organ formation, and exercising outer-secretion function human parotid glands cells could have or not when compounded with scaffold materials, so as to establish further foundation to construct the salivary glands of tissue engineering.Objective: To study the composites of collagen and chitosan made by cryogenic induced phase separation (CIPS) which have various volumes ration, suitable objectivebapertur , aperture rate, degeneration rate in vitro and vivo, and have good biocompatibility with HSG cells. The human parotid gland cells could be growth and organ formation, after compounded with the composite of collagen and chitosan and then implanted into nude mice. Therefore, the study would be to establish the further foundation to construct the salivary glands of tissue engineering.Method:1.Chitosan and composites of collagen and chitosan were prepared by cryogenic induced phase separation(CIPS).The surface form was observed by SEM and the objectivebapertur , then the aperture rate, and degeneration rate in vitro and vivo were calculated.2. To study the biocompatibility of HSG cells, the seed cells for tissue engineering of salivary glands, with chitosan and composite of collagen and chitosan for scaffolds choice of salivary glands, including the proliferation activity of HSG cells in the extract fluids of composite of collagen and chitosan, the proliferation activity of HSG cells when seeded on composite of collagen and chitosan, PLGA, PLGA/collagen. The condition of HSG cells on the composite was observed by SEM methods.3. HPGCs(human parotid gland cells) were cultured in vitro treated with Isoleucine at various ascending concentrations to findthe optimum concentration for growth and proliferation of HPGCs. The effect of Isoleucine on growth and proliferation of human parotid glands cells (HPGCs) at optimum concentration of Isoleucine was studied.4. The human parotid gland cells were cultured in vitro and combined with the composite of collagen and chitosan to form the compound artificial parotid gland. The compound artificial parotid gland were implanted into nude mice to observe the formation of parotid gland tissue by HE staining and to investigate the maintenance of their phenotypic and functional characteristics by immunohistochemical analyses. Results:1. Four various volumes rationed composites of collagen and chitosan made by CIPS were 1:1, 1:3, 1:9, and the chitosan was used as control. The objectivebapertur and aperture rate of chitosan were found to be120-200μm and 63.4% Observed by SEM, respectively, 120-400μm and 78.2% at the ration of 1:1; 120-400μm and 93.6% at the ration of 1:3; 80-200μm and 95.1% at the ration of 1:9.2.In the action of collagenase I, the degeneration rates of composites increased consistent with increasingly collagen contents while the Chitosan degenerated very slower in vitro.The same condition occurred when implanted in vivo, composite the ration was 1:9 has degenerated completely while the ration was 1:1 and 1:3 has degenerated about a half in 4w.3. The growth and proliferation activity of HSG cells in the every extracted fluids concentrations were in normal with the time prolonging. There was no significant difference in statistics. The materials have no toxicity.4. HSG cells could grow and proliferate on the surface of the composite of collagen and chitosan scaffold, and attach to the surface like corpuscle, gather to mass, grow into the...