Preparation And Application Of The Alginate Microcapsule In The Liver Tissue Engineering

Acute liver failure (ALF) as a high incidence of severe clinical liver disease, it has rapid progression, high mortality rate. Currently the most effective treatment of ALF is orthotopic liver transplantation (OLT), but the main problem of liver transplantation is facing the lack of donor, high cost of surgery and the treatment, and the need to take immunosuppressants for life. So there are still some clinical limitations. Some studies have used liver cell transplantation to replace orthotopic liver transplantation in the treatment of ALF, which has made some progress. But the liver cells have a high degree of environmental dependency for survival. After implantation, the host environment changed meantime had immune rejection and other factors, so the activity of cells decreased rapidly. Leading to treatment is not satisfactory. Therefore, the urgent need new treatments to improve the therapeutic effect of ALF.With the rapid development of tissue engineering, tissue engineering applied to more and more the treatment of liver disease, liver tissue engineering emerged. The ultimate goal is through artificial means in vitro cultivation as a normal metabolic function of liver tissue or organ type, in vivo transplantation in patients with impaired liver function after liver repair, rehabilitation, reconstruction until the full replacement. In recent years, the rapid development of liver tissue engineering around the seeding cell type, cell source, scaffolds and liver tissues and organs building systems in vitro– bio-artificial liver and bioreactor. There have been a large number of research studies domestic and foreign.A major problem of liver tissue engineering is the source of liver cells. It is difficult to amply autologous liver cells in vitro in a large number, although exogenous liver cells can be greatly received, but can not avoid the host immune rejection of the role of the damage. Cell microencapsulation technology which can package a variety of cells effectively protected, to some extent in vivo after transplantation to avoid immune rejection induced; the same time allowing all kinds of small molecule nutrients, free access to capsule, supporting cells proliferation and maintain their normal physiological functions. This technique effectively extends the liver tissue engineering in the scope of available seeding cells, the source of the cells from the liver cells by the expansion of liver cells to allogeneic source. Therefore, microencapsulated hepatocyte-based bioartificial liver of liver tissue engineering has become an important part.Alginate-polylysine-alginate (A-PLL-A) preparation method is by far the most in-depth research work and microencapsulation technique is widely used in various types of cells parcels. Microencapsulated hepatocytes has achieved considerable progress, especially microencapsulated hepatocytes in acute liver failure and so on. Nevertheless, preparation of alginate microcapsules there are limitations and problems of its own, mainly in the physical stability and biocompatibility of microcapsules less so. The main result of these problems is not yet available microcapsules prepared materials to long-term stability in the clinical treatment of functional requirements. Therefore, to find and develop new types of microcapsules prepared material is microencapsulated artificial cell research, and is one of the main research directions and focus.Microencapsulated material is the basis of merits and demerits of microencapsulated cell performance, the researchers by increasing the alginate purity of the chemical composition of alginate microcapsules performance to search for new alternative materials and other methods to obtain physical and biological properties, such as better compatibility, more stable and reliable microencapsulated materials, and carried out extensive research. Alginate was purified by the research, found that high purity of the alginate microcapsules can improve the biocompatibility and mechanical strength; by Ba²⁺ replaced Ca ²⁺ produced a barium alginate Poly- L-lysine-alginate microcapsules (B-PLL-A), effectively improved the mechanical strength of microcapsules and physical stability; by poly-lysine (PLL) study found that alginate due to its combination of efficiency and lack of microcapsule membrane can lead to adhesion of fibroblasts and macrophages produce several cytokines, thereby reducing the long-term stability and biocompatibility of microcapsules. Therefore, the search for alternative materials PLL is also a hotspot.Poly-ornithine (PLO) is a surface with a positively charged amino acids. Recent studies show that, PLO combined with alginate, a result of the formation of chemical bonds between the two relatively short. Such replacing PLL with the PLO microcapsules were prepared material that can be increased the material stability to some extent. Currently, research is still in its infancy and focused on the APA microcapsules for reconstructed, and only reported in the literature corresponding to individual microencapsulated myoblasts in vitro evaluation of experiments. For BPA and other vesicles, with PLO instead of PLL, has not been reported.Our group has completed a preliminary Ba²⁺ replaced Ca²⁺ in the preparation of B-PLL-A microcapsules, and its physical properties and biocompatibility were evaluated. On this basis, this study developed PLO alternative PLL on the two alginate microcapsules (APA, BPA microcapsules) to explore the PLO on the physical properties and biocompatibility of alginate microcapsules. In vitro, by human liver HL-7702 cells were cultured microencapsulated preliminary evaluation of both liver cells microencapsulated meet and maintain the effectiveness of their normal physiological functions; then poly ornithine-based alginate microencapsulated mouse liver cells and implanted into the rat model of acute liver failure in vivo, studies based on this microencapsulation method of liver cell transplantation for acute liver failure results. In order to develop a more excellent performance of microencapsulated materials. Meanwhile, through microencapsulation technology to improve liver cell transplantation for treatment of liver diseases provide experimental basis.This dissertation includes the following four parts:Part I: the development of alginate microcapsules on poly-ornithine and the optimization of preparation conditionsAs a new type of microcapsule preparation material, PLO microcapsules didn't prepare in this laboratory platform had any experience. It is necessary to carry out its exploration and optimization of preparation conditions. This study used the following means to explore the preparation process: the preparation of microcapsules by microencapsulation voltage electrostatic apparatus occurred, injection pump speed, the needle diameter and alginate concentration, concentration and time of poly-amino acids (PLL, PLO) and many other changes in preparation conditions, investigated the conditions on the system of microcapsules, and optimized the preparation conditions.This study developed two new alginate-based PLO microcapsules: alginate-poly ornithine-alginate (A-PLO-A) microcapsules and barium alginate-poly bird acid-alginate (B-PLO-A) microcapsules.The results show that the optimization of the A-PLO-A, B-PLO-A microcapsule preparation conditions are: electrostatic field for the 6 6.5kV/1.5cm, pump speed of 15mL/h, inner diameter of needle 0.25mm, the concentration of sodium alginate 2%, concentration of PLO 0.05 0.10%, reaction time 8min, sodium citrate liquefaction time 8min. With the traditional A-PLL-A, B-PLL-A compared to optimal preparation conditions, electrostatic field voltage range and allows concentration of poly amino acids are more relaxative, which is in favour of changing conditions by adjusting the capsule system. Without reducing the quality of case microcapsules we could control the size of microcapsules. Optimization of preparation conditions of microcapsules for further evaluation of their physical properties and biological description of the foundation. At home and abroad have not see B-PLO-A reports on the successful development of microcapsulesPart II: Evaluation of physical and biological properties Based on poly-ornithine barium alginate microcapsulesThis part of the research on the B-PLO-A microcapsules with B-PLL-A microcapsule permeability environment stability, mechanical strength and permeability and other physical properties were tested, and their biological properties were evaluated.The results showed that: B-PLO-A microcapsules than B-PLL-A microcapsules more stable environment in low permeability, not easy to deformation damage; 14 days in vitro B-PLO-A microcapsules than B-PLL-A microcapsules diameter increased to a lesser extent; mechanical vibration after 96h B-PLO-A microcapsule integrity was 99.3±1.0%, B-PLL-A microcapsules was 96.2±1.5%, showed that new microcapsules in the original B-PLL- A microcapsule based on the mechanical strength has been further increased. The permeability of the microcapsules, B-PLO-A microcapsules and B-PLL-A microcapsules were able to 4kD free diffusion of small molecules into the microcapsules inside, 70kD middle-molecular substances may be in part through the capsule; the 150 kD macromolecular material is not easy by microcapsules membrane, showed that the two types of microcapsule membranes with appropriate molecular retention capacity. 8 weeks after intraperitoneal transplantation, removal from the rat peritoneal cavity B-PLO-A microcapsules with B-PLL-A microcapsule structure were more comprehensive, integrity the former was 97.3%±2.1%, the latter 95.4%±2.4%, two kinds of microcapsules were cylindrical and spherical, smooth surface, no cell package, no significant fibrosis, indicating B-PLO-A microcapsules with B-PLL-A microcapsules possess good biocompatibility. The corresponding reports at home and abroad are not been seen.Part III: Poly-ornithine on alginate microencapsulated human normal liver cell line in vitroIn this study, the first part of the optimization of the microcapsule preparation conditions on A-PLO-A and B-PLO-A two microencapsulated the HL-7702 cells in vitro morphology, metabolite concentration levels of detection, preliminary investigation liver cells in poly-ornithine microcapsule on the growth and proliferation in the case, in order to further explore the two new microencapsulated hepatocyte transplantation for the foundation.The results show that: A-PLO-A and B-PLO-A after two microencapsulated hepatocytes in vitro 1 week, both within the microcapsules can be formed in the microcapsules of different size and shape of the liver cell aggregates, micro- intracapsular cell number and volume growth than the initial package; transplantation 4w, recall found no significant deformation of microcapsules, cells surrounding and fibrosis the phenomenon. And the two kinds microencapsulated HL-7702 cells both had the continued ability of synthesis of albumin and urea, the concentration level had no significant difference. That the presence of microcapsule membrane did not have adherent contact inhibition of growth and characteristics of the HL-7702 cell proliferation and biological function, significant adverse effects. Thus this proved that poly-ornithine on the new alginate microcapsules met the needs of he survival of microencapsulated hepatocytes to some extent. And they may also maintain cells'normal physiological functions. Study of based on PLO microcapsules alginate and cultured in vitro liver cells has not been reported at home and abroad. Part IV: Poly-ornithine on alginate microencapsulated hepatocytes in vivo transplantation in the treatment of liver failureThis study investigated the implantation of A-PLO-A and B-PLO-A two new types of microencapsulated mice hepatocytes into rats with acute liver failure prepared by intraperitoneal injection of D-galactosamine. We observed rats survival, detection of microcystin in liver cell growth and proliferation, metabolism and whether or not improve the survival rate of liver failure in rats. The main study groups were A-PLO-A blank microcapsules, B-PLO-A empty vesicles, free of liver cell transplantation, A-PLO-A microencapsulated hepatocytes and B-PLO-A-microencapsulated hepatocytes five experimental groups.The results showed that only the empty microcapsules to liver failure in rats the role of illness without remission after transplantation. The transplantation group two empty capsule 3d mortality reached 100%. Free liver cell group, A-PLO-A group of microencapsulated hepatocytes and B-PLO-A group of microencapsulated rat hepatocytes ALT, AST, ALB levels of both resume, 4w microencapsulated transplantation group of the time free liver cells are better than standard group, indicating liver cell transplantation of microencapsulated effectively alleviate the extent of liver injury, the treatment effect was better than free liver cell group. Microencapsulated hepatocytes survival rate of rats was significantly higher than empty capsule group and the free liver transplantation in cell transplantation group; 4 weeks after the recovery of microcapsules smooth surface, no cell package, no fibrosis. Description based on poly-ornithine alginate microcapsules physical stability and biocompatibility, suitable as a carrier of liver cells in vivo. Report on PLO alginate microencapsulated hepatocyte transplantation for acute liver failure in vitro has not yet to be seen.In summary, this study successfully prepared two kinds of poly-ornithine new microcapsules A-PLO-A and B-PLO-A microcapsules and their preparation conditions were optimized. The study found PLO on the change of preparation conditions weaker than the sensitivity of PLL, so that the microcapsules in a more general electrostatic field pressure and the concentration of poly-amino acids to maintain the stability of microencapsulated quality, benefit by adjusting preparation conditions, to reach the different preparation conditions and flexible so as to obtain even better performances microcapsules; B-PLO-A stability in the physical and mechanical performance are further improved than B-PLL-A microcapsules, and with the appropriate retention capacity of the molecular and biological good compatibility; in vitro A-PLO-A and B-PLO-A microencapsulated HL-7702 cells to maintain normal biological characteristics, to further expand A-PLO-A and B-PLO-A microencapsulated mouse liver cell transplantation for treatment of liver failure in rats provides an experimental basis. Finally, A-PLO-A and B-PLO-A implantation of microencapsulated hepatocytes in rats with acute liver failure in vivo experiments showed that the A-PLO-A and B-PLO-A two kinds of microcapsules with the more obvious the treatment of acute liver failure capacity, these types of microcapsules for the further application of liver tissue engineering research lay a solid foundation.