The Experiment Research On Reconstituting Tissue Engineering Submandibular Gland In Vivo

ObjectiveExtreme salivary hypofunction is a common side effect caused by the autoimmune exocrinopathy Sj grens syndrome and the irradiation damage that occurs during therapy for head and neck cancer. Radiation therapy for malignant head and neck tumors is mainly responsible for inadvertent damage of the salivary glands. If the therapeutic radiation treatment exceeds ~ 50 Gy, generally irreversible damage occurs to the salivary glands. Xerostomia is the major symptom of this condition, with consequent mucositis, dental caries, dysphagia and nutritional deficits. Dry eye syndrome (DES) refers to a spectrum of ocular surface diseases with diverse and frequently multiple aetiologies. At present there is no suitable therapy for these patients, and they suffer from considerable morbidity. Xerostomia and its consequences can only be treated in a symptomatic way. Since 1994, Prof. Wang yuxin have successfully used microvascula submandible gland tansfer method to reconstruct lacrimal gland to treat DES. But this method is not well to the patients with salivary hypofunction. Following the development of cell biology and materiallogy, it provide a new experimental concept for reconstituting salivary gland function with culturing bioactive salivary gland cells in, vitro and planting in vivo with scaffold. Based on the principles of tissue engineering , application of this technology to treat the salivary gland diseases is very important and prospective in the future.Domestic and abroad scholars have done some basic works about tissue engineering of salivary gland. In our earlier studies, we have established an idealand effective cultivation system including the methods of isolation, purification and cultivation of salivary gland cell in vitro and investigate collagen sponge as the scaffold. The ultimum purpose of this study is to put the bioactive cells into scaffold then culture them in vivo and maintenance their proliferation and differentiation to establish an artificial salivary organ. At present there is no report for establish an artificial salivary organ with cell and scaffold combined culture in vivo.The purpose of this study is to establish an ideal and effective tissue - engineered salivary gland cultivation system including investigating the effects of Laiminin, Collagen I and TGF - β3 on growth behavior and secretion function of cultured cells in vitro and assessed the growth state, inflammatory responses, and general systemic response characteristics of optimization cells combined culture on a collagen sponge scaffold in vivo for such an artificial gland.Methods1. Role of- extracellular matrix (laminin and collagen I ) of rat submandibular gland cells ( RSGCs) culture in vitro1. 1 Isolated culture and seed of RSGCs : The whole submandibular glands were isolated and dissected.Submandibular gland cells were obtained and purified by direct separation and pancreatic enzyme digestion from the gland of the rat. Then, epithelial cells were cultured and subcultured in 10% fetal bovine serum on DMEM. The second - generation cells were used for experiment.1.2 The growth behavior and cell cycle examination of RSGCs: Observe proliferation and morphological behavior of cultured RSGCs with phase contrast microscope, acting growing cells were detached by 0. 25% trypsin and fixed with 4℃ ethanol for 30 minutes in suspension, washed 3 tines with ice cold PBS, treated with RNAase 1mg/ml for 30 minutes in 37℃ , centrifuged down, resuspended in 10mg/ml propidium iodide for 15 minutes in dark 4℃, then passed through flow cytometer.1. 3 Identification of cells origin: cells were stain for cytokeratin ( CK) , S -100 protein and Vimentin with immnohistochemistry to identify origin of cells. 2. The role of TGF - β3 on the amylase secretion of RSGCs 2. 1 The proliferation behavior examination of RSGCs with TGF - β3; RSGCs were cultured with different concentrations of TGF - β3 ranging from 0.5 to 25. 0ng/ml. MTT colorimetric method was used to estimate vital force of RSGCs after culturing 72 hours.2. 2 The amylase secretion examination of RSGCs with TGF - β3: Extracting the supernatants of culture fluid for 24,48,72 and 96 hours, Amylase protein was assayed by auto - biochemistry equipment and Western blot.3. Experimental study on in vivo implantation of allogenous RSGCs combined with collagen sponge in rat3. 1 Implantation of allogenous RSGCs combined with collagen sponge in vivo : RSGCs labeled by Brdu were seeded on collagen sponge (2cm × 2cm × 2cm). They were divided into three groups according to inserts (group A: RSGCs seeded on collagen sponge; group B; collagen sponge; group C: sham -operated controls ). Inserts of group A and B were implanted beneath the skin on the back.3.2 The growth behavior of RSGCs and immunological responses examination in vivo; At 0, 3, 5,7and 15 days postimplantation, implant sites were examined local and systemic responses were assessed by conventional clinical chemistry and hematology analyses. After paraffin embedding, serial sections 6 mm thick were cut and stained with either hematoxylin and eosin or brdu tissue stain for immunohistochemical studies and examined the responses of tissue. Scanning electron microscope was used to observe the growth behavior of RSGCs on collagen sponge scaffolds.Results1. Role of extracellular matrix (laminin and collagen I ) of rat submandibular gland cells ( RSGCs) culture in vitro1. 1 The growth behavior of RSGCs: The shape of cell was observed with phase contrast microscope as spherical shape when it was just seeded. After 8hours, the RSGCs of groups which laminin in the culture medium attached to the bottom and became flat. After 96 hours the cells became fused together and the shapes like road metal. The cells without laminin in the medium did not attach until cultured 24 hours after seeding.1. 2 The growth curve of RSGCs: At the first day the cells proliferated slowly. This period showed as incubation period corresponding to curve. The second day after seeding, the RSGCs with laminin in medium proliferated obviously, cell number increased quickly and came into exponential fashion until day 4. thereafter, cell number plateaued as growth ceased. The growth curve of the other groups moved to right and cell number increased slowly over the first 4 days and then in rapid, exponential phase of growth began.1. 3 Identification of cells origin; Culture cells were positive stained by CK8. 13 and S - 100 antibody. The CK8. 13 and S - 100 antibodies were epithelium specific and their staining showed strong positive. While the culture cells were negative stained by vimentin antibody, which was fibroblast specific.1.4 The cell cycle examination of RSGCs: flow cytometer analysis shows that compared with control group, the percent of cells in S + G2 + M phase which coated with laminin increased obviously ( P < 0. 01 ) but the cells in G0 + G1 phase decreased. In the collagen Ⅰ group, the percent of cells in S + G2 + M phase increased slightly ( P =0. 096).2. The role of TGF - β3 on the amylase secretion of RSGCs2. 1 Morphology observation; culturing with TGF - β3 (0. 5ng/ml ~10. 0ng/ml)in the medium, RSGCs became spherical functional form. There are many secretory granules in the cytoplasm. Under the transmission electron microscope , Microvilli, plasm crease and zymogen granules could be seen on the surface of acinus epithelial cells. Mitochondria and rough endoplasmic reticulum could be seen in cytoplasm.2.2 MTT colorimetric method estimated vital force of culture cells shows; there is no significant different among the groups (P >0.05).2.3 The amylase secretion examination of RSGCs; After 72 hours, compared with the control, TGF - β3 (0. 5ng/ml -10. 0ng/ml) significantly stimulated the amylase secretion of RSGCs. But high concentration of TGF - β3 (25.0ng/ml) showed no stimulation. Western blotting demonstrated RSGCs and submandibular gland has the same band of amylase electrophoresis.3. Experimental study on in vivo implantation of allogenous RSGCs combined with collagen sponge in rat3.1 General observation: All animals recovered readily from the implantation procedure. In group A, at the 3rd and 5th day after in vivo implantation, capsule formed in the surface of insert and thickened gradually. The insert became more and more adhesive to ambient tissue. Fifteen days after implantation, the capsule became integrity and gloss and capillary network can be seen. The contact surface between insert and subcutaneous tissue is smooth. In group B, it is similar to group A at the 3rd and 5th day, but at the 15th day after implantation the insert is not completely in shape.3.2 Observation by scanning electron microscope: After implantation invi-vo, the RSGCs could proliferate and synthesize collagen. In earlier , RSGCs presence in disorder at the first 3 days and attach to the collagen sponge at the 5' day. At the 15' day RSGCs proliferate on the collagen sponge scaffold and form functional unit.3. 3 Histologic observation; postoperative inflammatory reaction of either group A or group B is strong until the 7th day. In group A, the fibers of collagen sponge always arranged regularity, RSGCs proliferate on the collagen sponge scaffold fused to form functional unit 15 days after implantation. The results of labelled cells were positive by immunohistochemical methods at each time point. In group B, the fibers of collagen sponge arrangement became irragulary at the 15th day after operation.3.4 Immunological rejection observation; Blood samples from rats at each time point were pooled together according to each experimental group to provide sufficient sample volume for analyses. For most analyses, there were no major differences seen between the sham - operated and implant groups for any of the measures. Neutrophil levels were elevated initially postimplantation of implant groups, above those seen in the shamoperated rat, but returned to similar values by 15 days. Lymph cell levels have no significance change.