ObjectiveTo explore the degradation of AZ31 magnesium alloy and poly (lactic-co-glycolic acid) (PLGA) in the femoral condyle, and then evaluate the laws of degradation of AZ31 magnesium alloy by Micro-CT images and data. MethodsForty 3-month-old male New Zealand white rabbits (weighing, 2.5 kg) were randomly divided into 4 groups, 10 rabbits each group. Forty micro-arc-oxidized AZ31 magnesium alloy pins and 40 PLGA pins were implanted into the right and left femoral condyle, respectively. Micro-CT images and data analysis were used to evaluate the degradation at 4, 8, 12, and 16 weeks after operation (n=10). Degradation was evaluated by weight difference between pre-and post-implantation. The inflammatory response was observed around the implants by HE staining. The weight loss of magnesium alloy and Micro-CT results were compared. ResultsThe Micro-CT images showed that PLGA pins had gray low signal, which was similar to the soft tissue around. At 4 weeks after operation, no signs of degradation were observed, and there were little corrosion pitting on the magnesium alloy. At 8 weeks, corrosion pitting gradually expanded, the boundary between the longitudinal axis and the cross section became blurred; at 16 weeks, corrosion pitting became bigger, and the boundary was discontinuous. Micro-CT quantitative analysis showed that the volume fraction of magnesium pins decreased slowly at 4 and 8 weeks; it was significantly lower at 12 and 16 weeks than 4 and 8 weeks (P < 0.05). The magnesium cylinder mineral density continuously decreased during the study period, it had a rapidly speed from 12 to 16 weeks (P < 0.05). However, the magnesium CT image density showed a slight change (P>0.05). The surface-to-volume ratio of the pins constantly increased, and the ratio was significantly larger at 12 and 16 weeks than 4 and 8 weeks, and at 16 weeks than 12 weeks (P < 0.05). There was more and more corrosion pitting on the surface with time, which resulted in a decrease in the radius that mean trabecular thickness gradually decreased, showing significant difference between different time points after 8 weeks (P < 0.05). The weight loss detection showed that the degradation of magnesium pin and PLGA gradually increased with time (P < 0.05), and the degradation rate of magnesium pin was significantly lower than that of PLGA at 8-12 weeks (P < 0.05), but the degradation rate of magnesium pin was higher than that of PLGA at 16 weeks. At each time point, the weight loss of magnesium alloy was similar to that by Micro-CT, but mass fraction was lower than volume fraction and had significant differences at 8, 12, and 16 weeks (P < 0.05). HE staining revealed that slight inflammatory response was observed around the magnesium pins at 4 weeks, and inflammatory reaction gradually reduced with time and disappeared at 16 weeks, but no inflammatory reaction was seen around PLGA. ConclusionMicro-CT has the advantages of non-trauma, in vivo detection, quantitative analysis, and precise data in evaluating the degradation of AZ31 magnesium alloy. Regarding the degradation of the magnesium alloy and PLGA in vivo, the degradation rate is slow in the early stage, and then increases with time. The degradation of PLGA is faster and earlier but it is then overtaken by AZ31 magnesium alloy at 16 weeks. During the degradation, the density of the magnesium has almost no change. The biomaterials can not firmly attach to the surrounding tissues due to inadequate holding forces.
【Abstract】 Objective To develop a novel cartilage acellular matrix (CACM) scaffold and to investigate its performance for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-l inked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-β1, canine BMSCs were seeded into the scaffold. Cell prol iferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed that there are no chondrocytefragments in the scaffolds and that toluidine blue, safranin O and anti-collagen II immunohistochemistry staining werepositive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter (155 ± 34) μm, 91.3% ± 2.0% porosity and 2 451% ± 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or ell iptic morphology with much matrix secretion. Conclusion The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibil ity, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.
ObjectiveTo introduce a technique of frozen sections for undecalcified bone and discuss its feasibility by observing the fluorescence distribution of the bone and cartilage. MethodsThe male Sprague Dawley transgenic rats at the age of 8 weeks, which express green fluorescent protein were selected to isolate the whole knee sectioned by the undecalcified bone frozen section technique. Under the fluorescence and light microscopy, the fluorescence and structure were observed within the organization of slice. Immunohistochemical staining (collagen type Ⅰ and Ⅱ), HE staining, toluidine blue staining, and Alizarin red staining were performed to observe the distribution of fluorescent substance and cartilage and bone structure. ResultsThe thickness of sections prepared by this technology was 6 μm. General observation showed that the structure of sectioned joint was complete. Under the light microscope, the morphology of cartilage cells, the arrangement of subchondral bone, and trabecular bone traveling could be clearly distinguished. Under fluorescence microscope, green fluorescence was shown in the joint soft tissue, cartilage tissue, and bone tissue; collagen type Ⅰ expressed in the bone tissue, collagen type Ⅱ in cartilage tissue. HE staining and toluidine blue staining could clearly distinguish the morphology of the cartilage layer. Alizarin red staining showed the structural integrity of subchondral bone plate and the organization within the meniscus, and proximal tibia cortical bone continuity. ConclusionThe fluorescence distribution can be directly observe in the bone and cartilage by sectioning of frozen undecalcified bone. This new technology can shorten the cycle of preparing sections.