| Objective 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 osteogeneticdifferentiation of rabbit bone marrow mesenchyme stem cells (BMSCs).Methods Chitosan(CS) was mixed with β-glycerophosphate(C/GP) according to theproportion 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 proliferationin vitro was made to frozen section and stained with HE to observe the adhesion andgrowth of cells. The cell growth and proliferation of BMSCs was observed by CCK8assayand fluorescent cell live/dead assay.Results A combination of BMSCs／C／Gp could be solidified to to form the hydrogelunder the condition of37℃for10mins. Cultured in C/GP hydrogel extracts for14d, cellsproliferation quantity and displayed a normal, spread and polygonal morphology. Frozensection HE dyeing shows that C/GP hydrogel present homogeneous red dye and poresample structure. BMSCs distribution is more even, form for most of the round, the nucleusblue dye, a round or oval. CCK8assay showed significant difference in the experimentalgroup in the2nd day compared to the control group (P <0.05), but there are no significant difference in the4th,6th,8thday (P>0.05). Cell live/dead staining assay showed aftercultured for14d, live cells in the test group, stained green and displayed a normal, spreadand 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 closeto the body temperature conditions. C/Gp eat sensitive hydrogel was no cell toxicity toBMSCs and suitable for cell adhesion and grow, and laid the foundation for further testing. Objective 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 wasmade 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 solidpowder for making big pore. The phosphate buffer was used as the cement liquid. The CPCsolid 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 thenovel CPC material extraction was observed by CCK8assay. Scanning electron microscopywas used to observe material hole diameter and cell adhesion, growth in the material. Theexperiment 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. Thehole diameter value of the traditional CPC material reached6.66±2.58μm. There issignificant difference between the two group (p<0.05). The microporosity of the novel CPCmaterial 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). Maximumload, flexural strength and toughness of the novel CPC material are increased about1timesthan the traditional CPC (p <0.05). CCK8assay showed there are no significant differenceof the light absorption valuein of cells in the CPC extraction in the2th,4th,6th,8thdaycompare to the negative control group (p <0.05). The novel CPC materials cell toxicitywas 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 tissueengineering scaffold. Objective To research the protection of the heat sensitive hydrogel to the BMSCs packagein the process of the CPC self-solidifying and the change of adhesion, proliferation andosteoblast differentiation of BMSCs in the calcium phosphate cement including C/Gphydrogel encapsulating BMSCs and mannitol crystal. Methods Liquid C/GP solution containing MSCs was placed at the bottom of cell cultureplat and allowed to set at37°C to form hydrogel. The CPC paste was placed on the top ofeach hydrolgel disc and allowed to set at37°C to solidified. The cell growth andproliferation of BMSCs encapsulated in the C/Gp hydrogel in the process of the CPCself-solidifying was observed by CCK8assay and fluorescent cell live/dead assay. Alkalinephosphatase activity was observed by the organization chemical method. Alizarin redstaining assay was uesd to detect the expression of calcified nodule. RT-PCR was used todetect ALP and calcitonin mRNA expressions. Scanning electron microscopy was used toobserve cell adhesion and growth in the novel CPC material.Results After BMSCs cultured in C/GP hydrolgel-CPC for14d, the fluorescent celllive/dead assay showed the percentage of live cells and cell density was similar to thecontrols (p>0.05). For example, the percentage of live cells at14d was (78.77±2.66)%inC/GP hydrolgel-CPC,(82.07±4.30)%in signal MSCs suspension and (80.03±3.08)%inC/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. AfterBMSCs cultured with osteogenesis medium in C/GP hydrolgel-CPC for7d and14d, boththe ALP and alizarin red staining are positive and there is no difference with the controlgroup(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 bonemineral-like nano-apatite crystals after an encapsuled MSC cultured for5d on the novelCPC composite scaffold materials.Conclusion The heat sensitive C/GP hydrogel have the protection to the BMSCs duringthe process of the CPC self-solidifying. The novel CPC composite scaffold materials wasno cell toxicity. The three-dimensional pore structure and material properties of the novelCPC composite scaffold materials was suitable for BMSCs’ adhesion, growth andosteoblast proliferation. Objective To research the fabrication of Rabbit radial defect model and observe the novelCPC material’s repair effect to the rabbit radial defect model.Methods12adult health New Zealand big white rabbit were choosed and their middle ofradius were exposed and intercepted about1cm along their bilateral radial forearms tofabricate bone defect model. Six of them for the trial group were implanted new CPC, theother six for the control group implanted traditional CPC. The X-ray examination on thedouble upper limbs was made on the surgey day and4weeks,8weeks and12weeks aftersurgery. 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 andexclusion when implanted in the rabbit radial defect area. The X-ray examination showedfracture repair effect in the trial group was better than control on4weeks,8weeks and12weeks after surgery. The postoperative12weeks spiral CT3D reconstruction radial defectarea and visible surrounding normal bone integration complete, bone creeping substitutionideal, fracture defect clearance fuzzy disappear. Radial defect area of control groupimplanted traditional CPC material degradation absorption, fracture defect still exist, andnormal surrounding bone no obvious connection integration. After operated12weeks,histochemical assay showed the osteogenesis and bone moulding ability in the experimentalgroup 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 therewas no significant difference in Elastic modulus parameters (P>0.05).Conclusion The novel CPC material has good biocompatibility, biodegradability and cellactivity, to the rabbit model of radial defect repair has good curative effect, as the ideal forbone tissue engineering scaffolds have bright prospects. |
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