Application Of Porous Scaffold Poly(L-Glutamic Acid)/Chitosan For Adipose Tissue Engineering And Tumor Reserch

Part1Application of porous scaffold poly (L-glutamic acid)/chitosan on adipose tissue engineeringObjectiveLarge soft-tissue defects from traumatic injury, oncologic resection and congenital deformities do not repair spontaneously. The defects and resulting contour deformities lead to abnormal cosmesis, affect the emotional well-being of patients, and may impair function (e.g., limb range of motion). Current standard treatments primarily involve tissue transplantation, including composite tissue flaps and synthetic substitutes. However, limitations are donor-site morbidity, unpredictable outcomes due to graft resorption over time (40%-60%volume loss), allergic reactions, and fibrous capsule contraction, as well as complications and costly surgery.The development of adipose tissue engineering has made it an attractive approach with great potential for repairing large soft-tissue defects. The basic strategy of adipose tissue engineering involves the utilization of artificial of natural extracellular matrix as scaffolds in combination with specific types of cells under the stimulation of growth factors to restore defects structurally and functionally. The key points in adipose tissue engineering were composed with the selection of cell, biomaterials with biocompatibility and biodegradation as well as the microenvironment for growth and differentiation of cell.In this study, we constructed tissue-engineered adipose with adipose tissue-derived adult stem cells and a novel porous scaffold poly (L-glutamic acid)/chitosan (PLGA/CS) which was improved with electrostatic interaction between carboxyl groups of water soluble PLGA and amido groups of CS using the phase separation method. Besides, we investigated the biological characteristics of PLGA/CS and the attractive potential of PLGA/CS as scaffold for tissue engineering.Methods1. The adipogenic differentiation potential of ADSCs(1) We harvested ADSCs from fresh human lipoaspirates obtained from patients who had undergone abdominal liposuction with enzyme digestion method. After the standard culture in vitro, passage2cells were used in the study.(2) The expression of cluster differentiation (CD) markers on ADSCs was revealed by flow cytometry, including CD13、CD14、CD34、CD44、CD45、CD49d、CD90、 CD105, CD106and CD166.(3) Multi-lineage differentiation of ADSCs was reveal by ALP staining, the expression of collagen type11and Oil-red O staining.(4) The changes of morphologic features and intracellular lipid vacuoles were observed by light microscope and Oil-red O staining respectively.2. The adipogenic differentiation of ADSCs on PLGA/CS in vitro(1) PLGA/CS was prepared with PLA microspheres (diameter200-300μm) as porogens using freeze drying and static electricity method.(2) Scanning electron microscope (SEM) observation and fluorescent DiL labeling were carried out to reveal the attachment and growth of ADSCs on PLGA/CS.(3) The quantitative assay of cell proliferation with time was detected by DNA assay.(4) The cytotoxicity of PLGA/CS was revealed by the quantity assay for the expression of lactate dehydrogenase (LDH).(5) Oil-red O staining was carried out to assess the intracellular lipid accumulation of ADSCs on PLGA/CS with or without adipogenic induction. (6) RT-PCR was carried out to reveal the expression of adipocyte-specific and insulin-regulated genes, including peroxisome proliferatior-activated receptor y2(PPARy2), fatty acid-binding protein (aP2), lipoprotein lipase (LPL) and adiponectin of ADSCs on PLGA/CS with or without adipogenic induction.3. In vivo adipose tissue formationAfter2weeks of exposure to adipogenic medium in vitro, ADSC-seeded scaffolds were implanted into the dorsum of severe combined immunodeficient (SCID) mice (6weeks old); acellular scaffolds were a control (n=6each group). After6weeks, animals were sacrificed and implants were harvested. To examine whether volume-stable adipose tissue could be harvested, tissue volumes were measured by immersing the tissue in PBS in a graded pipette. The volume change of implants was the ratio of the volume of implants retrieved at6weeks to the initial volume. Furthermore, to demonstrate newly formed fat tissue, the constructs retrieved in both groups were flash-frozen in Tissue-Tek OCT freezing medium for Oil-red O staining.Results1. In2D culture, ADSCs isolated from fresh human lipoaspirates showed uniform fibroblast-like morphologic features. Flow cytometry revealed the positive expression of CD13, CD34, CD44, CD90, CD105, CD166, CD49d and negative expression of CD14, CD45and CD31; Multi-lineage differentiation of ADSCs was confirmed by ALP staining, the expression of collagen type Ⅱ and Oil-red O staining. Under non-differentiation culture, ADSCs retained their fibroblast-like spindle shapes, with negative staining for Oil-red O at7and14days. On incubation with adipogenic medium, cells resembled adipocytes， with a rounded appearance and a time-dependent increase in intracellular lipid vacuoles, with positive staining for Oil-red O at7and14days. Increased staining for Oil-red O demonstrated increased adipogenic differentiation of ADSCs with increased exposure time. After1and2weeks, Oil-red O staining was higher for differentiated ADSCs than controls (p<0.05).2. The PLGA/CS scaffold appeared as a soft and porous spongy-like mass. The porosity of this scaffold was greater than90%. SEM demonstrated the even distribution and abundance of ECM deposition surrounding PLGA/CS scaffolds at7days after seeding. Quantitative Hoechst33258assay revealed greatly increased number of cell-seeded constructs in both groups. Oil-red O staining was greater for cell-seeded scaffolds exposed to adipogenic medium for21days than non-induced constructs (p<0.05). RT-PCR revealed increased expression of adipocyte-specific and insulin-regulated genes.3. Six weeks after implantation, gross inspection revealed intact and well-vascularized cell-seeded scaffolds. Moreover, cell-scaffold constructs appeared to shrink, which had decreased to90.5%and82.3%of the original volume in the cell-seeded and acellular groups, respectively (p>0.05). Oil-red O staining demonstrated adipose tissue formation within adipogenic-differentiated cell-seeded scaffolds after6weeks’ implantation, with little adipose tissue formed within acellular implants.Conclusion1. ADSCs are an ideal autologous cell source for adipose tissue engineering.2. The PLGA/CS scaffold had good biocompatibility and biodegradation for cell adhesion and proliferation for tissue engineering, and it could be a promising novel scaffold material for tissue engineering.3. Even though the adipose-like tissue was confirmed in vivo study, a further study is needed to fabricate porous scaffolds with optimal ration of PLGA and CS which can match the kinetics of new adipose formation in vivo.OriginalityWe introduced a novel porous scaffold poly (L-glutamic acid)/chitosan (PLGA/CS) which was improved with electrostatic interaction between carboxyl groups of water soluble PLGA and amido groups of CS using the phase separation method and finished the first use of such scaffold for adipose tissue engineering. Part2Application of porous scaffold poly (L-glutamic acid)/chitosan for tumor3D-cultured modelObjectiveCholangiocarcinoma (CC) is the second most common from of primary liver cancer after hepatocellular carcinoma. CC is classified anatomically into extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC). Despite advances in diagnosis and treatment, the prognosis of CC has not yet been resolved, because it is difficult to make an early diagnosis and standard therapy is not very effective. CC is generally characterized by strong proliferation, invasion, and early metastasis. To improve the prognosis, we require a fuller understanding of the molecular mechanisms behind its proliferation and progression.Epithelial-mesenchymal transition (EMT) is an intricate process by which epithelial cells lose their epithelial characteristics and acquire a mesenchymal-like phenotype. This dramatic phenotypic changes involve the loss of E-cadherin-mediated cell-cell adhesion and additional prototypic epithelial markers, as well as the loss of apical-basal polarity, concomitantly with the acquisition of a motile behavior and a profound reorganization of the cytoskeleton. EMT was related to early embryogenesis, organ fibrosis as well as the process of tumor progression including invasion, metastasis and therapeutic resistance. EMT provides tumor cells with the ability to dissociate from each other and degrade and to actively migrate into the basal membrane and invade the adjacent connective tissues. ECM has been proved to relate to the tumor progression such as breast, prostate, ovarian, lung cancer. A great number of environment factors and signals have been described that induce EMT. Among the signaling pathways, transforming growth factor-(3(TGF-β) is probably the best characterized inducer of EMT in development, cancer and other pathologies.In this study, we first investigated the effect of TGF-β1on EMT of HCCC9810cell in2D culture model to provide the target for gene therapy. Besides, we translated tissue engineered scaffold PLGA/CS into3D cancer-cultured model to reveal the effect of such3D model on the growth and invasion of cancer.Methods1. In standard monolayer cultures, HCCC9810was cultured within induced medium composed with10ng/ml TGF-β1,1%FBS and RPMI1640, the cells cultured in growth medium was served as control group; the expected morphological changes were detected by direct microscopic observation. The simple wound-healing migration assay was carried out to assess the motility behavior of cells. The organization at cellular levels of the individual EMT markers (i.e. Vimentin, Fibronectin for mesenchymal marker; E-cadherin, β-catenin and Pan-CK for epithelial marker) was revealed by immunofluorescence assay, while the protein and mRNA levels were detected by WB and real-time PCR respectively.2. HCCC9810cells were seeded onto porous scaffold PLGA/CS. Scanning electron microscope (SEM) observation and fluorescent DiL labeling were carried out to reveal the attachment and growth of cells on PLGA/CS; the quantitative assay of cell proliferation with time was detected by DNA assay. The mRNA levels of EMT-related markers and TGF-β1was detected by real-time PCR.Results1. Under microscopic observation, the cells in induced group exhibited the loss of cell-cell contacts and cobblestone morphology, appearance of elongated mesenchymal features along with growth as individual cells; the wound healing assay showed the motility behaviror of cells cultured in induced medium. Immunophenotypical characterization in2D cultures showed the expression of β-catenin transformed from the membrane into cytoplasm; no expression of E-cadherin; down-regulated expression of Pan-CK; the higher expression of mesenchymal markers fibronectin and vimentin after induction; F-actin cytoskeleton reorganization was observed. Real-time PCR showed down-regulated expression of E-cadherin and β-catenin; the up-regulated expression of fibronectin, vimentin and N-cadherin. WB showed no expression of E-cadherin; the less expression of β-catenin; the higher expression of fibronectin, vimentin and N-cadherin which were similar to the results of IF and real-time PCR.2. SEM demonstrated the even distribution and abundance of ECM deposition surrounding PLGA/CS scaffolds. Quantitative Hoechst33258assay revealed greatly increased number of cell-seeded constructs, whereas the proliferation rate is less than that of2D cultured cell. The results from3D model reflected the real growth state of tumor in vivo. Real-time PCR showed the expression of E-cadherin and β-catenin was significantly decreased after21days post seeding (p<0.05); the expression of vimentin and fibronectin was significantly increased after14and21days post seeding (p<0.05); interestingly, the expression of TGF-β1was increased after14and21days post seeding (p<0.05).Conclusion1. We confirmed that HCCC9810cells underwent EMT process induced by TGF-β1. The expected morphological changes and the expected changes in EMT-related markers do occur at the cellular, protein and mRNA level.2. We translated tissue engineering technology platforms into cancer research and provided more physiological models for cancer research. We found such scaffold-based3D culture model could promote the autocrine of TGF-β1to improve the EMT and tumor invasion.Originality1. TGF-β1may become the target for gene therapy of cholangiocarcinoma.2. Tissue-engineered scaffold PLGA/CS was first utilized for cancer research, and the3D culture model could be promising tool for tumor invasion and metastasis as well as the development of therapeutic drugs.