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Fabrication of large-sized bone tissue in vivo by injectable differentiation-control modules and cell-culture modules

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Journal Title:
Chinese Journal of Biomedical Engineering
Key Word:
骨形态发生蛋白;模块化;组织工程;可注射性;骨和骨组织;分化微控单元;细胞单元;大尺寸;Bone morphogenetic protein;Modularity;Tissue engineering;Injectability;Bone and bones;Differentiation-control modules;Cell-culture modules;Large size

Abstract´╝Ü Objective:To explore a simple and practical method for fabricating large-sized bone tissue in vivo.Methods:Bone marrow mesenchymal stem cells (BMSCs) were primarily cultured in bilateral femurs and tibias of rats. The surface markers of BMSCs were identified by flow cytometry. The Cell morphology was observed by inverted microscope.The osteogenic differentiation of BMSCs was observed by Von Kossa mineralized nodule staining, and the adipogenic differentiation of BMSCs was observed by oil red O staining. The differentiation-control modules were constructed by binding CBD-BMP2 (bone morphogenetic protein-2 containing collagen-binding domains) to micron-scale collagen filaments, and the cell-culture modules were constructed using rat bone marrow mesenchymal stem cells (BMSCs) and Cultispher-s gelatin beads. Then, differentiation-control modules and cell-culture modules mixture (experimental group) , cell-culture modules alone (control group 1) , and differentiation-control modules alone (control group 2) , contained in a syringe, were injected subcutaneously into the back of 11 male SD male rats, respectively. The rats in the three groups were sacrificed after being raised for 30 days. The biomimetic tissues cultured subcutaneously wereharvested. Electron microscope and micro-CT scanning were used to observe the surface and internal structure of the biomimetic tissues. HE staining and alkaline phosphatase staining of the biomimetic tissue were used to observe the distribution of cells and the stained expression of osteogenic componentsin the tissues. Alkaline phosphatase activity in the biomimetic tissues was examined using 5-bromo-4-chloro-3-indole-phosphate (BCIP) /Nitrobluetetrazolium (NBT) alkaline phosphatase chromogenic kit. Energy spectrum analysis was used to determine the level of calcium ions in the biomimetic tissues. Compressive modulus was used to measure the biomechanical strength of the biomimetic tissues.Results:The expression rates of CD44 and CD90, the surface positive antigens of P3 generation BMSCs, were 95.11% and 96.18%, respectively.The proportions of hematopoietic surface markers CD11b and CD45 were 1.18% and 1.82%, respectively. BMSCs are spindle shaped, fusing into sheets and swirling. After osteogenesis induction of BMSCs, a large number of black mineralized nodules were formed. A large number of round red lipid droplets were formed after adipogenesis induction of BMSCs. The bone-like tissues cultured were approximately 1.3 cm × 2.1 cm × 0.6 cm in size with a fairly firm texture in the experimental group, and 1.0 cm × 1.0 cm × 0.4 cm in size with a soft texture in the control group 1. No formed tissues were cultured in the control group 2. Electron microscope and Micro-CT scanning showed that the biomimetic tissues in the experimental group presented a bony structure consisting of a surface with high signal intensity and an internalspace massively filled high signal intensity of bony components, but in the control group 1, no structure with high signal intensity was found in the biomimetic tissue. HE staining showed formation of bony tissues massively in the experimental group, but not the least in the control groups. The experimental group showed more production of alkaline phosphatase than did the control group 1, as well, the alkaline phosphatase activity was higher in the experimental group than that in the control group 1 [ (0.023±0.005) nmol·mg -1·min -1 vs (0.005±0.002) nmol·mg -1·min -1, P<0.05]. The percentage content of calcium ion in the experimental group was 7.42% vs 0.34% in the control group 1. The compressive modulus was higher in the experimental group than that in the control group 1 [ (2.62±1.41) MPa vs (0.03±0.01) MPa, P<0.05]. Conclusion:Fabrication of large-sized bone tissue in vivo by differentiation-control modules and cell-culture modules can be simple and feasible. The injectability of cell-culture modules and differentiation-control modules allows infusion of these modules into bone fracture spaces or defects for therapeutic purpose, and therefore provides a new ideation for introducing tissue engineering into clinical trials.

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