Construction Of CPC Bone Tissue Engineering Scaffold Mixing BMSCs-encapsulating Chitosan Hydrogel And Experiments Of Treatment Of Bone Defect

Part Ⅰ Research of chitosan/β-glycerol phosphate hydrogel encapsulating bone marrow mesenchyme stem cellsObjective To study the production of heat sensitive chitosan/β-glycerol phosphate (C/GP) hydrogel and the influence as the vector on the adhesion, proliferation and osteogenetic differentiation of rabbit bone marrow mesenchyme stem cells (BMSCs).Methods Chitosan(CS) was mixed with β-glycerophosphate(C/GP) according to the proportion of3:1and was placed under the condition of37℃to form the solid hydrogel. The mixture of C/GP hydrogel and rabbit BMSCs which has been cultured and proliferation in vitro was made to frozen section and stained with HE to observe the adhesion and growth of cells. The cell growth and proliferation of BMSCs was observed by CCK8assay and fluorescent cell live/dead assay.Results A combination of BMSCs/C/Gp could be solidified to to form the hydrogel under the condition of37℃for10mins. Cultured in C/GP hydrogel extracts for14d, cells proliferation quantity and displayed a normal, spread and polygonal morphology. Frozen section HE dyeing shows that C/GP hydrogel present homogeneous red dye and pore sample structure. BMSCs distribution is more even, form for most of the round, the nucleus blue dye, a round or oval. CCK8assay showed significant difference in the experimental group in the2nd day compared to the control group (P<0.05), but there are no significant difference in the4th,6th,8th day (P>0.05). Cell live/dead staining assay showed after cultured for14d, live cells in the test group, stained green and displayed a normal, spread and polygonal morphology similar to the controls. Dead cells are stained red and very few. The percentage of live cells and cell density was similar to the controls (p>0.05).Conclusions C/Gp heat sensitive hydrogel can keep for a long time hydrogel state in close to the body temperature conditions. C/Gp eat sensitive hydrogel was no cell toxicity to BMSCs and suitable for cell adhesion and grow, and laid the foundation for further testing. Part II Research of the novel calcium phosphate cement production and it's cell toxicity, mechanical properties and pore structureObjective To investigate the cell toxicity of a novel macropores calcium phosphate cement (CPC) scaffold and its influence on cell adhesion, growth and proliferation.Methods In the Novel CPC solid phase of the mixing process, tetracalcium phosphate [TTCP:Ca4(PO4)2O] and dicalcium phosphate anhydrous (DCPA:CaHPO4) powder was made a mixture of the proportion of the CPC solid powder according to1:1(Moore than). The mass ratio of50%of the water-soluble mannitol crystals was added to the CPC solid powder for making big pore. The phosphate buffer was used as the cement liquid. The CPC solid powder and curing liquid were mixed in the port according to the proportion of2g:1ml and get paste mixture, namely the CPC dough. The cell growth and proliferation in the novel CPC material extraction was observed by CCK8assay. Scanning electron microscopy was used to observe material hole diameter and cell adhesion, growth in the material. The experiment of three point bending was used to test the biomechanics performance of the novel CPC material.Results The hole diameter value of the novel CPC material reached267.43±118.01μm. The hole diameter value of the traditional CPC material reached6.66±2.58μm. There is significant difference between the two group (p<0.05). The microporosity of the novel CPC material is66.15±6.91% and the microporosity of the traditional CPC material is35.02±4.71%. There is significant difference between the two group (p<0.05). Maximum load, flexural strength and toughness of the novel CPC material are increased about1times than the traditional CPC (p<0.05). CCK8assay showed there are no significant difference of the light absorption valuein of cells in the CPC extraction in the2th,4th,6th,8th day compare to the negative control group (p<0.05). The novel CPC materials cell toxicity was rated grade1, namely the material on the BMSCs no significant toxicity.Conclusion The novel CPC material has the strong biomechanics performance, macropores, high microporosity and excellent biocompatibility, which is promising for ideal bone tissue engineering scaffold. Part Ⅲ Research of cell activity and osteogenesis differentiation in the novel Calcium phosphate cement including C/Gp hydrogel encapsulating BMSCs and mannitol crystalObjective To research the protection of the heat sensitive hydrogel to the BMSCs package in the process of the CPC self-solidifying and the change of adhesion, proliferation and osteoblast differentiation of BMSCs in the calcium phosphate cement including C/Gp hydrogel encapsulating BMSCs and mannitol crystal. Methods Liquid C/GP solution containing MSCs was placed at the bottom of cell culture plat and allowed to set at37℃to form hydrogel. The CPC paste was placed on the top of each hydrolgel disc and allowed to set at37℃to solidified. The cell growth and proliferation of BMSCs encapsulated in the C/Gp hydrogel in the process of the CPC self-solidifying was observed by CCK8assay and fluorescent cell live/dead assay. Alkaline phosphatase activity was observed by the organization chemical method. Alizarin red staining assay was uesd to detect the expression of calcified nodule. RT-PCR was used to detect ALP and calcitonin mRNA expressions. Scanning electron microscopy was used to observe cell adhesion and growth in the novel CPC material.Results After BMSCs cultured in C/GP hydrolgel-CPC for14d, the fluorescent cell live/dead assay showed the percentage of live cells and cell density was similar to the controls (p>0.05). For example, the percentage of live cells at14d was (78.77±2.66)% in C/GP hydrolgel-CPC,(82.07±4.30)% in signal MSCs suspension and (80.03±3.08)% in C/GP hydrogel. The live cell density at14d was (82.54±4.17)% in C/GP hydrolgel-CPC,(86.37±4.81)% in signal MSCs suspension and (83.63±5.20)% in C/GP hydrogel. After BMSCs cultured with osteogenesis medium in C/GP hydrolgel-CPC for7d and14d, both the ALP and alizarin red staining are positive and there is no difference with the control group(p>0.05). RT-PCR showed the expression of ALP and CT mRNA gene was intensified. The SEM shows BMSCs had resided into a pore in the composition and anchor to the bone mineral-like nano-apatite crystals after an encapsuled MSC cultured for5d on the novel CPC composite scaffold materials.Conclusion The heat sensitive C/GP hydrogel have the protection to the BMSCs during the process of the CPC self-solidifying. The novel CPC composite scaffold materials was no cell toxicity. The three-dimensional pore structure and material properties of the novel CPC composite scaffold materials was suitable for BMSCs' adhesion, growth and osteoblast proliferation. Part Ⅳ Fabrication of Rabbit radial defect model and the treatment effect of the novel CPC materials on fracture defect modelsObjective To research the fabrication of Rabbit radial defect model and observe the novel CPC material's repair effect to the rabbit radial defect model.Methods12adult health New Zealand big white rabbit were choosed and their middle of radius were exposed and intercepted about1cm along their bilateral radial forearms to fabricate bone defect model. Six of them for the trial group were implanted new CPC, the other six for the control group implanted traditional CPC. The X-ray examination on the double upper limbs was made on the surgey day and4weeks,8weeks and12weeks after surgery. On12weeks after surgery, the MRI examination on the double upper limbs, biopsy HE dyeing observation and biomechanics determination was made.Results The novel CPC material did not appear adverse reactions such as inflammatory and exclusion when implanted in the rabbit radial defect area. The X-ray examination showed fracture repair effect in the trial group was better than control on4weeks,8weeks and12weeks after surgery. The postoperative12weeks spiral CT3D reconstruction radial defect area and visible surrounding normal bone integration complete, bone creeping substitution ideal, fracture defect clearance fuzzy disappear. Radial defect area of control group implanted traditional CPC material degradation absorption, fracture defect still exist, and normal surrounding bone no obvious connection integration. After operated12weeks, histochemical assay showed the osteogenesis and bone moulding ability in the experimental group were obviously better than that of control group. After operated12weeks, biomechanics measurement showed significant difference of the Fmax, Flexural strength and F/d parameters in the experimental group compared to the control group (P<0.05), but there was no significant difference in Elastic modulus parameters (P>0.05).Conclusion The novel CPC material has good biocompatibility, biodegradability and cell activity, to the rabbit model of radial defect repair has good curative effect, as the ideal for bone tissue engineering scaffolds have bright prospects.