Development of Tissue Scaffold UsingLaster Sintering and Evaluation of Hydrogel Coating In Vitro Stand of Chondrocytes

• Main Authors: Zhi Hong Tang, 唐智宏
• Other Authors: M. Y.Lee
• Format: Others
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/85689116303190042461

碩士 === 長庚大學 === 醫療機電工程研究所 === 97 === Cartilage is a connective tissue, in the absence of the distribution of blood vessels and nerves, nutrient delivery and waste to be discharged to extracellular matrix by diffusion, therefore, cell regeneration and repair of cartilage slow; such as articular cartilage injury due to self-repair capacity poor, often have to rely on surgery to repair cartilage or artificial joint replacement to fill in order to avoid the deterioration for the sequelae of degenerative arthritis. The commonly used methods to repair cartilage tissue repair, including law, law tissue transplantation, cell implantation and so on, as a result of these methods would have to be the auto-or allograft cartilage implant to complete, cartilage implants, however due to difficult access, and are often immune rejection after implantation, so the use of tissue engineering technology to produce biodegradable stent implants, through in vitro cell proliferation in vivo experiments to generate cartilage implants, has become the present-day research in the field of regenerative medicine of the main topics. In addition the use of tissue-engineered cartilage implants technical training required to build the first porous scaffold, the production of most of the current stent forming method using molds (for example, salting-out method, gas foaming method, lamination film, etc.), can not have complete control over the pore structure inside the stent (size, density) and the link between the holes, and the impact of in vivo generation of cartilage tissue and the quality of cartilage regeneration after implantation. In recent years, the literature pointed out that the use of polymer-coated plastic cell water has become a way of non-stent 』, successful cartilage repair tissue defects of the method. However, hyaluronic acid and gelatin with the water-binder polymer is a three-dimensional network structure of hydrophilic and high moisture content the temperature sensor liquid / solid phase change materials, living cells can be coated to provide in vitro proliferation of the environment, promote cartilage cartilage cells to divide and to avoid, and have a patch of the implant and defect, but the lack of structural support strength, unable to bear the forces applied to the cartilage defect, resulting in limited clinical application. In addition, articular cartilage has a soft / hard two-phase composite structure of bone, and therefore applies to the human tissue-engineered articular cartilage repair, it is important to be able to produce bio-compatible and has a soft / hard two-phase composite tissue scaffold of bone, and can function with cell proliferation for clinical application. Yuan, the purpose of this study cartilage biopsy in order to overcome the above-mentioned problems faced by generation, were the laser sintering of soft / hard two-phase composite tissue scaffold bone production, polymer water-binder holes three-dimensional scaffold perfusion experiments, the use of both water-bearing structure of the production of plastic stent support strength and cell proliferative activity of the cartilage tissue implants; the outside in order to verify the effectiveness of in vitro cell culture experiments and clinical application of the feasibility of this study was also carried out cell attachment, proliferation, such as experiments and customized ear cartilage cells in vivo experimental process planning and ear shape molding stent pre-assessment study. This research work is divided into three parts, the first major work for the design of functional cartilage tissue scaffold in vivo, mainly the use of laser sintering rapid prototyping systems to produce biodegradable materials have been poly caprolactone (PCL) three-dimensional holes stent. Stents have also been produced material properties analysis, giant three-dimensional scaffold structure / micro-morphological analysis, mechanical strength, compressive / tensile testing, bio-solution rate and reduce the porosity of the experiment, etc.; the second part of the main support for the multi-level system so water-binder polymer stent perfusion experiments and in vitro cell culture experiments. First of all, three different methods of reperfusion immersion (immersion), pinhole injection (injection), vertical drop method of gravity (gravitational dropping) irrigation water into the holes of plastic stents to assess the water-cement and the effectiveness of stents and holes, followed by White rabbit articular cartilage cells back to obtain training and the use of experiments to assess the rate of cell-attached cells in the scaffold of the attached rate, further analysis of in vitro cell culture experiments. In vitro cell culture experiments Design of experiments is divided into three groups analysis: control group (TCPS), the experimental A (scaffold plus cells) and experimental B (water-binder plus stent cell); experiment to 28 days for the Department of cycle every seven days out three groups of 10 samples for analysis of a series of experiments to understand the cell growth, as every seven days to be carried out in a series of experiments for analysis of cell viability (MTS), Analysis of glucosamine (GAG), collagen Analysis (Total Collagen), electron microscopy (SEM) experiments to analyze cells in polycaprolactone (PCL) scaffold pore effect on proliferation and differentiation; the work of the third part of the main ears customized cell culture cartilage in vivo experimental process planning and ear shape molding stent assessment, the work includes a combination of Planning, Rapid Prototyping and salting-out method stent tissue engineering bioreactor culture and animal experiments and the use of CAD-mimics the shape of outer ear mapping software type rapid prototyping machine with the production of silicone molds (upper and lower molds) such as pre-study. This study has been completed using the laser sintering rapid prototyping system to produce the stent with mechanical strength combined with good biocompatibility of the polymer with water-binder of the experiment, through their own experiments to prove that the production of laser sintering systems in the mechanical testing of cartilage scaffold There are very good elastic modulus of 3.75MPa, the more documentation that the elastic modulus of normal cartilage (0.79MPa) too high, can be used in the body by the force area, laser sintering stent clinical needs can also set its porosity and pore size cultured cells; in the multi-layered polymer stent system made of plastic water perfusion stent is confirmed by experiments in comparison to three types of experimental design, vertical drop method of gravity (gravitational dropping) longer than one hour into the stent completely uniform, and then combined with the high - plastic water molecules after the in vitro quantitative, qualitative experimental data by the statistical analysis also found that activity in the cell (MTS), the first 0 days for just the volume of injected cells, the experimental group in the three groups had no significant difference p> 0.05, to the first 7 days, the control group and experimental group AB statistical difference p <0.05, due to cells that have not yet adapted to growth in the stent group and the control (TCPS) is a linear growth, the first 14 days of the control group and experimental group B statistical difference p < 0.05, due to the experimental group B (stent + cell + water glue) cell activity in the lower section within 21 days of the control group and experimental group A statistical difference p <0.05, after 14 days in 2D due to no support when attached to the TCPS covered with cells, the lower cells can not absorb nutrients and to make the activity of apoptosis also declined, the first 28 days there are statistical differences between three groups, because of the control group after the cell covered with a linear decline in activity in the experimental group A and group B continued to grow, and in the experimental group B (stent + cell + water glue) in the active group than the reasons for the experimental group A as a result of cells that were not covered with plastic water touch nutrients, mass transfer rate slower in the test to be placed in 4 ℃ water soluble glue resulted in cell activity when cooling down, but the number of 21 days has increased significantly. In the analysis of glucosamine (GAG), in culture from 0 to 7 days, cell proliferation at this time also, the secretion of GAGs very scarce, so are no significant differences p> 0.05, to the first 14 to 28 days control group and AB experimental group had statistical difference p <0.05, within the cell because of the control group without stent附著poor growth also resulted in less secretion of GAGs, the experiment would be made aware of cells attached to stent cells can provide a better environment for the growth of. Analysis of the collagen protein (Total Collagen) in the control group and experimental group B in the 0 ~ 28 days have statistical difference p <0.05, due to the control group and experimental group in the experiment time A secreted collagen content is not more than Experimental Group B and it is continuing to grow and much higher than other groups, presumably as a result of water-binder containing hyaluronic acid (HA) and gelatin (Gelatin) cartilage can promote proliferation, we can see that the support plastic containing the water cells can provide a better environment for the growth of. SEM graph in cells can also be found in a circular pattern and, on behalf of cell growth in the stent in a perfect system, also confirmed the water with a plastic stent in the cartilage repair technology is feasible; also planning a third part a set of custom ear cartilage cells in vivo experimental procedures, improve processes related to research for the future. This study is to study cartilage repair, cartilage tissue by promoting self-repair capacity in order to maintain the function of cartilage due to the principle of the production of tissue-engineered cartilage for the repair required to do in order to reduce the burden of health care.