Repair Of Articular Cartilage Defects In Rabbits Using Porous Chitosan Scaffold Containing Microspheres Loaded With Transforming Growth Factor-β₁

PartⅠPreparation and evaluation of the characteristics of different deacetylated degree of chitosan scaffoldTo evaluate the effect of different deacetylated degree of chitosan scaffold, it was using the SEM to observe the morphology and the rate of porosity, evaluate the swelling of water absorption and degradation in vitro and in vivo test. The results showed that the different deacetylation of scaffold was highly porous and three-dimensional structure; with increasing of the deacetylated degree, theircorresponding porosity was 93.6%,90.0%,85.1%; the rate of swelling was 820%,803%,772%; On the fourth week in vitro, the degraded rates were 30.44%, 22.88%,17.10%; while, in vivo the corresponding rate were 57.48%,40.23%, 29.53%.The degraded rate of chitosan scaffold were negatively correlated to deacetylated degree, furthermore, it showed that the speed of degradation in vivo was faster than the level in vitro. By controlling deacetylated degree of chitosan (almost 80%). it would be a booming and suitable material for the reparation of cartilage defects.PartⅡThe preparation and detection of chitosan loaded with TGF-β1microspheresObjective:Using emulsified cross-linking methods to prepare the chitosan loaded with transforming growth factor-β1(TGF-β1) microspheres, it detected the characteristics of swelling ratio, loading drug and TGF-β1.releasing amount of chitosan, and further to assesse the feasibility of using biodegradable chitosan scaffold as a carrier for controlled release of TGF-β1.Methods:to take the Sorbitol Oleate Benzene-80 as the emulsifier and sodium tripolyphosphate (TPP) as cross-linking agent, it used emulsified cross-linked methods to prepare the chitosan microspheres. It was embedded TGF-β1-releasing or bovine serum albumin (BSA) microspheres. The surface of the specimens of chitosan were detected by means of Scanning Tunneling Microscopy, the microsphere diameter measurement by using the laser particle distribution method; The microsphere swelling rate, drug loading amount, entrapment efficiency and sustained releasing rate by vitro assay.Results:it found that the microspheres diameters were concentrated centripetally and evenly distributed, with an average particle size of 35μm, smoothly spherical surface; the swelling rate in the acidic environment was the highest, which was up to 800%; the encapsulated efficiency of them was 88%. the TGF-β1 loading amout was 11ng/mg, the cumulative releasing amount of TGF-β1 and BSA was 90%, 63.3% in the seventh day, respectively. The gradual depredated rate under the action of lysozyme was 57% in the sixth week.Conclusion:Chitosan loaded with microsphere was feasible and facilitated by cross-linking emulsion.The material of TGF-β1 microspheres embedded chitosan in cartilage engineering had potential applications, with good releasing properties.Part IIIBiocompatibility of sustained-releasing microspheres loading chitosan scaffolds Objective:to achieve a kind of chitosan microspheres scaffold which loaded with transforming growth factor-β1(TGF-β1) in a sustained-release manner, evaluated its biocompatibility in vivo and in vitro, providing experimental evidence that chitosan scaffolds would be a promising material to repair of cartilage defects.Methods:chitosan scaffolds of loading and sustained-releasing microspheres with porous structure were prepared using technique of cross-linking emulsion and phase separation.By using hemolytic test, that of acute systemic toxicity experiment, skin stimulus experiment, the heat source experiment and implantation in muscle test for comprehensive evaluation of biocompatibility.Results:The chitosan scaffolds had three-dimensional interconnected porous structure and its diameter was 200-350μm, whose holes were separate by plates, the rate of porosity was 93.63%±0.51%(n=6, X±S); hemolytic rate of scaffolds was 1.6%, while had no significant destruction of red blood cell via the microscope; the result of acute toxicity evaluation was non-toxic, the liquid to extracte from chitosan scaffolds immersing for 24h,48h,72h was intradermal injected into mice, whose weight correspondingly were 0.3467±0.1075,0.4020±0.0796 versus 0.4932±0.0838, paired T test compared each group with the control saline group, P> 0.05; the points of skin to the primary stimulation and the primary stimulation index (PII) are 0; in the heat experiment,the temperature rised by 0.17±0.06; rats from implantation in muscle test were all survived well without systemic infection, and appearance of newborn normal hair was in the 4 weeks; and in the 8 weeks Naked-eye observed that there was significantly increased peripheral vascular stents, which well integrated with the surrounding muscle tissue, while other internal organs such as heart, liver, lung and kidney were ordinary, furthermore, from the 1 week to 12 weeks, infiltration of lymphocytes was gradually decreased,blood vessels and fibrous tissues around the frame is visible, simultaneously, wrapped fibrous tissues were gradually thin, chitosan scaffolds were gradually degraded.Conclusion:chitosan scaffold loaded porous microspheres had excellent biocompatibility, good three-dimensional pore structure and biodegrade ability, all that induce it was expected to be a good repair material for the treatment of osteochondral defects. Part IVRepair of articular cartilage defects in rabbits using porous Chitosan Scaffold Containing Microspheres Loaded with Transforming Growth Factor-β1Objective:To investigate the effect of MS-TGFs homing bone marrow cells and the signal factor from the marrow microenvironment in vivo, inducing bone marrow cells differentiated and regenerated into functioned cells in the cartilage defects location.Methods:To prepare chitosan loaded with the sustained release of transforming growth factor-β1 microspheres and chitosan composite scaffold by cross-linking emulsion, liquid separation methods, respectively. The specimens were observed using a scanning electron microscopy SEM for the surface of microspheres and chitosan after being gold-coated with a sputter coater; the microsphere diameter by Measurement of laser particle distribution, ELISA sandwich method to measure the entrapment efficiency, drug loading and release rate of TGF-β1 microsphere and the degradation of microsphere and scaffold in the fourth week; the full-thickness articular artilage defect deep to subchondral bone of 4.2mm diameter and 7 mm depth was created by drilling, then to implant ate different materials [TGF-β1 microspheres/ chitosan scaffold (MS-TGFs), TGF-β1/chitosan scaffold (CS-TFG), pure chitosan scaffold (CS)], spacious blank was constitution of the fourth group, compared to observe the recovery effect. In the first month and third month after surgery, samples were harvested, respectively. The repair in general was graded according to the criteria reported previously as Masuoka score. The harvested tissue were fixed by toluidine blue, respectively, and then assessed by expression of COL II, and comprehensive assessment of the quality of tissue repair by Wakitani score.Results:The microspheres diameters were concentrated centripetally and evenly distributed, with an average particle size of 35μm, smoothly spherical surface, The encapsulated efficiency of them was 88%. The TGF-β1 loading drug was llng/mg, in the first 7 days, the cumulative release amount from the microspheres was about 63.3%.The degradation of post-implantation 4 weeks was 48.5%. Four groups have no joint cavity infection, effusion; the Masuoka score Of MS-TGFs, CS-TFG, CS, free group were 7.67±0.47; 3.83±0.75; 1.00±0.89; 0.830.75, respectively. Harvested tissue from the first and third month, TB staining in the MS-TGFs group showed the best repair, with smooth surface, neat rows in articular cartilage, the integrity and continuous in cell structural; CS-TFG was basically repaired, but the formation is poor, less cartilage; CS was filled with fibrous cartilage; free group without repair, the defect diameter was about 5mm; the first month tissues by CD34, CD44 double staining identified the homing cells from stem cells. The CD34 (-) CD44 (+) cells in the MS-TGFs group were the largest, following CS-TFG group. The results of toluidine blue Wakitani score 4.50±1.12; 10.83±0.37; 13.67±0.47 (p< 0.01).Conclusion:The salt-leached chitosan scaffolds can be used for various tissue-engineering applications that cartilage defects can be repaired by homing cells.