ObjectiveTo study the effect of three-dimensional (3D) printed β-tricalcium phosphate (β-TCP) scaffold loaded poly (lactide-co-glycolide) (PLGA) anti-tuberculosis drug sustained release microspheres on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its cytotoxicity.MethodsIsoniazid and rifampicin/PLGA sustained release microspheres were prepared by W/O/W multiple emulsion method. The β-TCP scaffolds were prepared by 3D printing technique. The microspheres were loaded on the scaffolds by centrifugal oscillation method to prepare composite materials. The BMSCs of Sprague Dawley rat were isolated and cultured by whole bone marrow adherent method, and the third generation cells were used for the following experiments. BMSCs were co-cultured with osteogenic induction medium (group A), PLGA anti-tuberculosis drug sustained release microsphere extract (group B), 3D printed β-TCP scaffold extract (group C), and 3D printed β-TCP scaffold loaded PLGA anti-tuberculosis drug sustained release microsphere composite extract (group D), respectively. Cytotoxicity was detected by cell counting kit 8 (CCK-8) method; the calcium deposition was observed by alizarin red staining; and the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were detected by real-time fluorescence quantitative PCR (RT-qPCR).ResultsCCK-8 assay showed that the absorbance (A) value of groups A, B, C, and D increased gradually with the culture time prolonging. After cultured for 24, 48, and 72 hours, the A value decreased in the order of groups A, C, B, and D. There was no significant difference between groups B and D (P>0.05), but there were significant differences between other groups (P<0.05). The cytotoxicity was evaluated as grade 0-2, and the toxicity test was qualified. Alizarin red staining showed that red mineralized nodules were formed in all groups at 21 days after osteogenic induction, but the number of mineralized nodules decreased sequentially in groups C, D, A, and B. RT-qPCR test results showed that the relative expressions of OCN and BSP genes in groups A, B, C, and D increased gradually with the culture time prolonging. The relative expression of ALP gene increased at 7 and 14 days, and decreased at 21 days. After cultured for 7, 14, and 21 days, the relative expressions of ALP, OCN, and BSP genes decreased sequentially in groups C, D, A, and B; the differences were significant between groups at different time points (P<0.05).Conclusion3D printed β-TCP loaded PLGA anti-tuberculosis drug sustained release microsphere composites have no obvious cytotoxicity to BMSCs, and can promote BMSCs to differentiate into osteoblasts to a certain extent.
ObjectiveTo clarify the structure and biomechanical characteristics of the dura mater of the cervical, thoracic, and lumbar segments of sheep, in order to provide a theoretical reference for the study of artificial dura mater.MethodsFive adult male white sheep were sacrificed. The dura mater of C5, T10, and L3 planes were obtained. The histological HE staining was used to observe the internal structure and the thickness of dura mater; the inner and outer surfaces morphology of the dura was observed by scanning electron microscopy (SEM); transmission electron microscopy (TEM) was used to observe the internal structure of dura mater and to measure the diameter of collagen fibers in each part of dura mater. The dura mater of C6, C7, T11, T12, L4, and L5 planes were taken for uniaxial biomechanical test, and modulus of elasticity, tensile strength, and elongation at break were measured.ResultsHE staining showed that the thickness of the cervical, thoracic, and lumbar dura mater gradually decreased, and the thickness of the dura mater was (268.19±15.91), (198.16±27.25), (103.74±21.54) μm, respectively, and the differences were significant (P<0.05). SEM observation showed that there were more collagen fibers and fewer cells on the inner surface of the dura mater, while more cells were distributed on the outer surface, and the cells on the inner and outer surface were stretched along the longitudinal axis. TEM observation showed that the collagen fibers in the dura mater were interlaced and arranged in layers. The collagen fibers in the lamina were arranged in the same direction, and the collagen fibers between the lamina were arranged vertically. The diameters of collagen fibers in the cervical, thoracic, and lumbar dura mater were (68.04±21.00), (64.54±20.64), (60.36±19.65) nm, respectively, and the differences were not significant (P>0.05). Uniaxial biomechanical tests results showed that there was no significant difference in modulus of elasticity, tensile strength, and elongation at break between the axial and transverse dura mater of the cervical dura mater (P>0.05); the axial data of thoracic and lumbar segments were significantly larger than the transverse data (P<0.05). The axial modulus of elasticity, tensile strength, and elongation at break of the dura mater of the cervical, thoracic, and lumbar dura mater were significantly different (P<0.05) from the transverse ones, and showing a decreasing trend. Among them, the ratio of axial and transverse modulus of elasticity of cervical and thoracic dura were significantly smaller than that of lumbar segment (P<0.05), and there was no significant difference between cervical segments and thoracic segments (P>0.05).ConclusionThe thickness of dura mater in sheep decreased gradually from head to tail. There are more collagen fibers and fewer cells on the inner surface of dura mater, while the outer surface of dura mater is covered by cells. The collagen fiberboard layers in the dura mater are arranged alternately, and have obvious anisotropic biomechanical characteristics, and the anisotropic biomechanical characteristics get more significant from the head to the tail.