ObjectiveTo investigate the appropriate concentration of methicillin-resistant Staphylococcus aureus (MRSA) in establishing chronic femoral osteomyelitis model in rabbits.MethodsForty-eight adult New Zealand white rabbits were randomly divided into 6 groups with 8 rabbits in each group. Animals in groups B, C, D, E, and F were injected 1×109, 1×108, 1×107, 1×106, 1×105 CFU/mL MRSA on the location of 2 cm of the femoral supracondyle, respectively, and group A was injected with aseptic saline as a control. The general observation were performed at 4 weeks after operation, and the wound secretions were taken for bacteriological examination. The serum C-reactive protein content was detected at preoperation and 2 weeks and 4 weeks after operation. The X-ray, CT scan, and Norden imaging scoring were performed at 4 weeks after operation. At 4 weeks after operation, the animals were sacrificed, and the specimens were observed and evaluated by general scores; and the HE staining and histological score were also performed.ResultsFive rabbits died of severe infection in group B, 2 died in group C, and no rabbit died in groups D, E, and F. General observation showed that the incision healed without soft tissue swelling in group A; most animals had visible incision swelling and sinus formation, femoral thickening, bone destruction, and damage decreased with the decreasing of the concentration of liquid bacterial in groups B-D; the infection signs were seen in groups E and F, and the degree of infection were less than that of group D. Bacteriological examination showed that fistula formation animal in groups B, C, D, and E were cultured with positive results, and with the decrease of concentration, the number of animal fistula formation decreased gradually; and bacteriological culture did not be performed in group F because of no sinus formation. There was no significant difference in the content of C-reactive protein between groups before operation (P>0.05). The contents of C-reactive protein in groups B-F were significantly higher than those in group A at 2 and 4 weeks after operation (P<0.05). At 4 weeks after operation, the content of C-reactive protein was in the order of groups B, C, D, E, F, and A in turn from high to low, showing significant differences between groups (P<0.05). Imaging examination showed that there was no soft tissue swelling and bone destruction in group A; bone destruction, massive sequestrum formation, and soft tissue swelling were found in groups B and C; bone destruction was observed in groups D and E, and the degree of sequestrum formation was not as good as that in group C; and there was a small amount of bone infection in group F. The Norden scores in groups B-F were significantly higher than that in group A, and in groups B and C than those in groups D, E, and F, and in groups D and E than that in group F (P<0.05); there was no significant difference between groups B and C, and between groups D and E (P>0.05). The specimens general observation scores in groups B-F were significantly higher than that in group A, while in groups B and C than those in groups D, E, and F (P<0.05); there was no significant difference between groups D, E, and F (P>0.05). HE staining showed that the structure of bone trabecula in group A was clear and the structure was arranged neatly; in groups B-F, trabecular bone destruction and inflammatory cell infiltration were seen and the degree gradually decreased. The histological scores in groups B-F were significantly higher than that in group A, and in group B than those in groups C-F, in groups C and D than that in group F (P<0.05); there was no significant difference between groups C, D, and E, and between groups E and F (P>0.05).ConclusionThe optimal MRSA concentration of rabbit model of chronic osteomyelitis of femur is between 1×106 and 1×107 CFU/mL.
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.