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Chapter 1 different deacetylated chitosan scaffold and degradation of performance evaluation Objective: To prepare different degree of deacetylation of chitosan scaffolds investigated its body outside degradation performance of chitosan as a cartilage defect repair scaffold to provide an initial experiment basis. Methods: with a degree of deacetylation of 65%, 80%, 95% chitosan, phase separation Preparation of three-dimensional scaffold, SEM to observe the surface morphology, pore size, containing lysozyme in the liquid substitution method detection porosity, water-absorbing swelling ratio; enzyme 107U / L of PBS solution (pH 7.4) 37 ° C air bath oscillation, measured at different times (1d, 3d, 7d, 14d, 21d, 28d) stent degradation rate of implanted the SD rats vertical spine intramuscular, were detected degradation rate of two weeks, four weeks, six weeks, eight weeks, 10 weeks, 12 weeks and observed degradable stent tissue. Results: different degree of deacetylation bracket with the three-dimensional structure of the high porosity, porosity increase with the degree of deacetylation 93.6%, 90.0%, 85.1% the stent in color from pale yellow gradient white swelling rate of 820%, 803%, 772%, in the role of in vitro lysozyme gradually degrade different degree of deacetylation 28d stents were degraded to 30.44%, 22.88%, 17.10%, and the gradual degradation of the degradation rate of 57.48% in the first four weeks, 40.23%, 29.53%. HE detected is shown having a good compatibility with the surrounding muscle tissue Conclusion: The stent has a good three-dimensional pore structure, is gradual degradation in vivo, the higher the degree of deacetylation of decomposing slower, faster in vitro to in vivo degradation rate of the same time point, degradation, deacetylation degree of 80% of degradation rate and eight weeks of normal cartilage repair consistent. Chapter 2 sustained release TGF-β, chitosan microspheres preparation and performance testing purposes: use of emulsifying crosslinked prepared chitosan microspheres parcel of transforming growth factor-β1 (TGF-β1), and detection of the swelling rate , drug loading and sustained release performance and to assess the use of chitosan microspheres as a controlled release TGF-β1 carrier feasibility. Methods: liquid paraffin emulsifier, sodium tripolyphosphate (TPP) as a crosslinking agent, emulsion cross-linking prepared chitosan microspheres. Wrapped TGF-β1 and TGF-β1 bovine serum albumin (BSA), were prepared chitosan microspheres with BSA chitosan microspheres. The application of scanning electron microscopy, laser particle size distribution measuring instrument detection microsphere morphology, detection ball swelling ratio, a comprehensive analysis of the ELISA sandwich method for the determination microsphere drug loading, encapsulation efficiency and in vitro drug release rate of microspheres characteristics. Results: Preparation of microsphere size distribution is concentrated, the average particle size of 35μm, spherical, smooth and uniform surface of the sphere, swelling in the acid environment the highest rate of 800%; 88% higher encapsulation efficiency, drug loading was 11ng/mg, drug release tests showed that TGF-β1 and BSA are slowly released from microspheres cumulative release amounted to 90%, and 63.3% in the first seven days; progressive degradation of lysozyme degradation, degraded to 6 weeks 57 percent. Conclusion: The prepared emulsion crosslinking chitosan microspheres method is simple, TGF-β1 obtained chitosan microspheres with good release properties, as cartilage tissue engineering materials with potential applications. Chapter 3 Load biodegradable controlled release microspheres of chitosan scaffolds Biocompatibility Research Objective: To prepare stent biocompatibility in vitro and in vivo evaluation of the load sustained release microspheres of chitosan for chitosan as a class of promising animal cartilage defect repair scaffolds provide experimental basis. Methods: load release microspheres porous chitosan scaffold prepared emulsion crosslinking method and phase separation technique. Hemolysis test, acute toxicity test, intradermal stimulation experiments, the heat source experiment, intramuscular implantation experiments, the overall evaluation of the biocompatibility of chitosan scaffolds homemade load release microspheres. Results: Material aperture multiple 200-350μm and the interconnected three-dimensional porous structure, each hole is separated from a plate, a porosity of 93.63% ± 0.51% (n = 6, x ± s); stent hemolysis rate was 1.6%, endoscopic no significant destruction of red blood cells; materials acute toxicity evaluation of the degree of non-toxic, materials, extracts of mice 24h, 48h, 72h changes in body weight is .3467 ± 0.1075,0.4020 ± 0.0796,0.4932 ± 0.0838, respectively, at each time point and physiology saline group group, paired t-test, P gt; 0.05; intradermal primary stimulus scoring and primary irritation index (PII) are 0; the pyogenic experiment body temperature rise height of 0.17 ± 0.06; intramuscular implantation of experimental rats survived general good, no infection, about four weeks newborn hair normal distribution, general observation eight weeks the bracket around blood vessels increased significantly, well-integrated with the surrounding muscle tissue, heart and liver and lung and kidney and other organs had no special, one week, two weeks, four weeks, eight weeks, 12 weeks, with time, gradually reduce the infiltration of lymphocytes, visible blood vessels and fibrous ingrowth bracket, wrapped gradual thinning of stents becoming degradation. Conclusion: Load Microspheres porous chitosan stent has excellent biocompatibility, having a good three-dimensional pore structure and biodegradable, and is expected to become a good cartilage repair material. Chapter 4 Preparation of Supported TGF-β1 microspheres chitosan scaffolds and studied its purpose: to develop a of load sustained release TGF-β1 microspheres chitosan scaffolds rabbit articular cartilage defect repair, explore its in vivo adsorption own marrow cavity in the bone marrow cells and signaling factors in the microenvironment induced cartilage defect in situ regeneration of cartilage cells differentiated effector. : Emulsifying crosslinked prepared chitosan microspheres for sustained release TGF-β1, separated from the liquid phase prepared chitosan scaffolds, fared composite scaffolds using environmental scanning electron microscopy (SEM) observation stand and microsphere morphology laser particle size distribution measuring instrument detection microsphere diameter distribution ELISA sandwich method micrometer ball TGF-β1 encapsulation efficiency, drug loading and sustained release rate in vitro detection of micro ball and bracket 4 weeks degradation rate; selection of rabbits as experimental animals caused bilateral femoral trochlear Ministry of full-thickness cartilage defects, the use of different materials [TGF-β1 microspheres / chitosan scaffolds (MS-TGFs) of TGF-β1 / chitosan scaffolds (CS-TFG), pure chitosan scaffolds (CS) implant defect situ defect, the blank group control (empty)] constitute four groups to observe the repair effect. After January, March sacrificed general observation of cartilage repair status and to Masuoka rated, fixed tissues stained with toluidine blue, collagen type II immunohistochemistry, and Wakitani score a comprehensive assessment of the quality of tissue repair. Results: The four groups of rabbit knee implants no joint cavity infection, the the effusion, Masuoka score the MS-TGFs, the CS the TGF-beta, CS, Empty group were 7.67 ± 0.47; 3.83 ± 0.75; 1.00 ± 0.89; 0.83 ± 0.75. Organization 1,3 TB staining drawn showing MS-TGFs repair best filled surface smooth articular cartilage lined cell structural integrity, continuous; CS-TGF repair poor surface smoothness, and less cartilage; CS for a large number of fibrocartilage tissue augmentation; the Empty group without repair, and surrounding cartilage secondary damage, defect diameter of about 5mm; January CD34 group identification of CD44 double adsorption cells as a source of stem cells. MS-TGFs group CD34 (-) CD44 () cells, followed by CS-TGF group. March toluidine blue and collagen type II immunohistochemistry staining obvious cartilage cells and collagen metachromatic organizations the repair quality Wakitani rated shows: 4.50 ± 1.12:10.83 ± 0.37; 13.67 ± 0.47, a statistically significant difference (P lt ; 0.01). Conclusion: chitosan composite scaffolds can be repaired to some extent, the non-weight-bearing area of ??articular cartilage defects can promote cell homing original induced chondrocyte differentiation, and repair of cartilage defects.
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