| Objective: Bone tissue engineering is the key to repairing the structure and function of bone defect. Three-dimensional printing(3D printing)technology in low temperature and vacuum freeze-dried technology had been applied to prepare bionic composite scaffolds in our experiment to make the bionic of morphological structure and biomechanics of bone tissue engineering material come true. Meanwhile, silk fibroin, collagen and nanometer hydroxyapatite as raw materials and MC3T3- E1 cells as seed cells had been used during the course of fabrication, which built functional unit and provided foundational theoretical basis for clinical treatment of bone defect.Methods: 1. The extraction of raw materials for bone tissue engineering scaffolds: we extracted silk fibroin by boiling, degumming of silk, dialysis and enrichment of mulberry silk, and prepared collagen by salting out method which concluding fresh bovine tendon with acetic acid solution containing pepsin. 2. The preparation of bionic composite bone tissue engineering scaffolds: we mixed silk fibroin, collagen, and nanometer hydroxyapatite with the mass ratio of 3:9:2, then put it into 24 orifice after fully mixed, which was kept overnight at- 80?. We used vacuum freeze-drying mechanism to prepare composite scaffolds(marked for group A). At the same time, we took silk fibroin, collagen, and nanometer hydroxyapatite with the same mass ratio into the 3D printer cylinder at low temperature after mixing well blended. We set 3D printing control parameters, used 3D printer in low temperature and vacuum freeze-dried mechanism to prepare composite scaffolds(marked for group B). Two groups of scaffolds were prepared by anhydrous ethanol, Na OH solution, sterilizing with Co60 and to reserve. 3. The detection of physical and chemical performance of the bionic bone scaffolds: the micro computed tomography scanning technology was used to analyze the scaffold structure of group A and B. Mechanical testing machine was applied todetect the performance of scaffolds. X-ray diffractometer, Fourier transform infrared(FTIR) spectrometer and differential scanning calorimeter were also used to test the molecular structure of the scaffolds. 4. The establishment of tissue engineering co-culture model: MC3T3-E1 cells of the same density were inoculated on the two scaffold groups and cultured for 4 hours, then added ?-MEM complete medium. Group A was marked as control group and group B was marked as experimental group. The culture solution was changed for every 2 days with equal liquor quantity. 5. The cytocompatibility of the bionic bone scaffolds: MTT and alkaline phosphatase(ALP) method were respectively used to detect cell proliferation and differentiation of control group and experimental group. Inverted microscope and scanning electron microscope(SEM) were utilized to observe the cells growth on the surface and internal of each scaffold group. Real-time PCR and Western blot method were applied to detect the expression level of MC3T3-E1 cells specific gene.Results: 1. The scaffolds remained stable in morphology of both groups, group B were more neat than group A and1 more complied with the model design. 2. Scaffold materials in the two groups were three-dimensional structures with porous, porosity and water absorption rate. The scaffold pore sizes in group A were(163.15 ± 61.93) ?m, thickness was(290.42 ± 71.19) ?m, porosity was(92.21 ± 2.16) %, and water absorption expansion rate was(724.09 ± 98.05) %; The macro-pore sizes in group B were(506.37 ± 18.63) ?m and micro-pore sizes were(62.14 ± 17.35) ?m, thickness was(91.63 ± 18.11) ?m, porosity was(97.70 ± 1.37) %, and water absorption expansion rate was(1341.97 ± 64.41) %. Elastic modulus of group A and B respectively were(31.91 ± 11.25) k Pa and(340.93 ± 71.98) k Pa, showing that mechanical property of group A was lower than group B(P < 0.05). Characteristic peaks of scaffold materials in group A and group B by X-ray diffractometer, FTIR and differential scanning calorimeter were similar, showing that there was no obvious change in protein molecular structures, molecular crystallinity and intermolecular forces. 3. Results determined by MTT method and the activity of ALP detection showedthat the cell proliferation and differentiation of experimental groups were significantly higher than the control groups. An intuitive view of the cell morphology was given by inverted microscope and scanning electron microscopy with the cells being well attached to the scaffolds and revealing both flattened and spindle shaped morphologies. MC3T3-E1 cells grew well on the macro-pore walls of experimental group and part of the macro-pores could be full of cells for 21 days cultured. Scaffolds and cells were co-cultured after 21 days, Real- time PCR results showed that the transcription levels of MC3T3-E1 cells COL?, ALP and OCN genes were significant up-regulation on experimental group. Western blot results showed that the protein translation level MC3T3-E1 cells COL?, ALP and OCN genes on both control group and experimental groups were satisfactory.Conclusion: 1. The morphology of two groups was neat with three-dimensional porous structures. 3D printing at the low temperature with personal parameter made the bone tissue engineering scaffolds controllable in pore sizes, which could provide more area for cell adhesion. The pore sizes, porosity, water absorption expansion rate and mechanical properties of Group B scaffolds were superior to group A, which was in favour of the transportation of cell nutrients and metabolites. 2. The combination of 3D printing with freeze-dried technology didn't destroy the molecular structures and protein activity of the materials and the scaffolds maintained the biologic activity of the organic and inorganic components. 3. The cellular proliferation and differentiation of the experimental group were more superior to the control group, which showed that the microstructures of scaffolds could affect its mechanical properties and biological behavior. The scaffolds prepared by the combination of 3D printing with freeze-dried technology could further realize the bone units to repair bone defects. |
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