ObjectiveTo evaluate the ectopic osteogenesis of recombinant human bone morphogenetic protein 2 (rhBMP-2) loaded chitosan (CS)/dextran sulfate (DS) by micro-CT. MethodsrhBMP-2/CS/DS microspheres were prepared by the ionic crosslinking and its shape was observed under the scanning electron microscope. The release of rhBMP-2 was determined from resultant microspheres by ELISA assay. Forty-eight Sprague Dawley male rats were randomly divided into 4 groups (n=12), quadriceps muscle bag model was made, gelatin sponge (group A), CS/DS microspheres (group B), rhBMP-2 (group C), and CS/DS/rhBMP-2 microspheres (group D) were implanted into the bags respectively. The tissue samples with heterotopic ossification were harvested for micro-CT scanning at 4, 8, 12, and 16 weeks. The tissue mineral density (TMD), bone volume fraction (BVF), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and tissue mineral content (TMC) were measured. ResultsThe prepared rhBMP-2/CS/DS microspheres with smooth surfaces were spherical and evenly disperses without obvious agglomeration. At 2 hours, microsphere started a sudden release period in vitro; the release reached a peak at 2 days; and the release cycle lasted about 20 days. The rats survived to the end of the experiment. At each time point after operation, no radiation developed and no osteogenesis was observed by three dimensional reconstruction in groups A and B. However, radioactive strength and reconstructed bone tissue gradually increased in groups C and D, and group D had more radioautography and more bone tissues than group C. At each time point, TMD, BVF, Tb.Th, Tb.N, BMD, and TMC of groups A and B were zero. Ectopic bone formed with time, the other parameters showed an increasing trend except Tb.N in groups C and D, showing significant difference when compared with groups A and B at each time point (P < 0.05). There was no significant difference between groups C and D at 4 weeks (P>0.05); the parameters of group D were significantly higher than those of group C at 8-16 weeks (P < 0.05). ConclusionrhBMP-2/CS/DS microspheres have stronger ability of ectopic bone formation than single rhBMP-2.
Objective To investigate the feasibility and application value of digital technology in establishing the micro-vessels model of cross-boundary perforator flap in rat. Methods Twenty 8-week-old female Sprague Dawley rats, weighing 280-300 g, were used to established micro-vessels model. The cross-boundary perforator flaps of 10 cm×3 cm in size were prepared at the dorsum of 20 rats; then the flaps were suturedin situ. Ten rats were randomly picked up at 3 and 7 days after operation in order to observe the necrosis of flap and measure the percentage of flap necrosis area; the lead-oxide gelatin solution was used for vessels perfusion; flaps were harvested and three-dimensional reconstruction of micro-vessel was performed after micro-CT scanning. Vascular volume and total length were measured via Matlable 7.0 software. Results The percentage of flap necrosis area at 3 days after operation was 19.08%±3.64%, which was significantly lower than that at 7 days (39.76%±3.76%;t=10.361, P=0.029). Three-dimensional reconstruction via the micro-CT clearly showed the morphological alteration of micro-vessel of the flap. At 3 days after operation, the vascular volume of the flap was (1 240.23±89.71) mm3 and the total length was (245.94±29.38) mm. At 7 days after operation, the vascular volume of the flap was (1 036.96±88.97) mm3 and the total length was (143.20±30.28) mm. There were significant differences in the vascular volume and the total length between different time points (t=5.088, P=0.000; t=7.701, P=0.000). Conclusion The digital technology can be applied to visually observe and objectively evaluate the morphological alteration of the micro-vessels of the flap, and provide technical support for the study of vascular model of flap.
Objective To realize the visualization of three-dimensional microstructure of rabbit sciatic nerve bundles by micro-CT and three-dimensional visualization software Mimics17.0. Methods The sciatic nerve tissues from 6 New Zealand rabbits were divided into 2 groups (n=3), and the sciatic nerve tissues were stained by 1% (group A) and 5% (group B) Lugol solution respectively. After staining for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 hours, the imaging changes of specimens were observed by light microscope and micro-CT. The clear micro-CT images were exported to the Mimics software to complete the visualization of three-dimensional microstructure of rabbit sciatic nerve according to three-dimensional reconstruction tool. Results The clear three-dimensional microstructure images could be observed in group A at 2.5 hours after staining and in group B at 1.5 hours after staining by light microscope and micro-CT. The sciatic nerve of New Zealand rabbits were divides into 3 bundles and each of them was relatively fixed. There was no obvious crossing or mergers between each bundle. The cross-sectional area of each bundle was (0.425±0.013), (0.038±0.007), and (0.242±0.026) mm2 respectively. The digital model could clearly reflect the microstructure of the sciatic nerve at all cross sections. Conclusion The internal structure of New Zealand rabbits sciatic nerve can be clearly reflected by micro-CT scanning. It provides a reliable method for establishing a nerve microstructure database with large amount specimens.
ObjectiveTo study the ectopic osteogenesis and biocompatibility of bone morphogenetic protein 2 (BMP-2)-derived peptide P24 loaded chitosan-4-thio-butylamidine (CS-TBA) hydrogel.MethodsFirst, the CS-TBA/hydroxyapatite (HA) solution was prepared by using chitosan, 2-iminothiolane hydrochloride, and HA. Then, the different amount of P24 peptides were added to the CS-TBA/HA to prepare the CS-TBA/5%P24/HA and CS-TBA/10%P24/HA solutions. Finally, β-glycerophosphate disodium (β-GP) was added to the CS-TBA/HA, CS-TBA/5%P24/HA, and CS-TBA/10%P24/HA to prepare the CS-TBA/HA/β-GP, CS-TBA/5%P24/HA/β-GP, and CS-TBA/10%P24/HA/β-GP hydrogels, respectively. Eighteen Sprague Dawley female rats were randomly divided into 3 groups (n=6), which were injected into the back muscle pouches with equal volume CS-TBA/HA/β-GP hydrogel (group A), CS-TBA/5%P24/HA/β-GP hydrogel (group B), and CS-TBA/10%P24/HA/β-GP hydrogel (group C). The animals were sacrificed at 4 and 8 weeks and conducted micro-CT. The ability of biodegradation and osteogenesis of hydrogl was detected by trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and histological staining (HE and Masson).ResultsAll the rats survived to the time point of the harvest. Micro-CT results showed that the new bones gradually increased in each group after operation. At the same time, the new bone formation was more obvious in groups B and C than in group A, and with the increase of P24 concentration, new bone formation in group C was much more than that in group B. The Tb.Th, Tb.N, and BMD increased gradually in 3 groups, and the differences between 4 and 8 weeks were significant (P<0.05) except the Tb.Th in group A. At different time points, the Tb.Th, Tb.N, and BMD were significantly higher in groups B and C than in group A (P<0.05), and in group C was higher than in group B (P<0.05), showing significant differences between groups. Histological staining showed that the materials of groups B and C were biodegradable, and the osteogenic effect was increased with the increase of P24 concentration.ConclusionP24 peptide can improve the ectopic osteogenesis of CS-TBA hydrogel, and the 10% concentration is more effective.