| Objective:GeneX is a new artificial bone which consists of a mix of β-tricalcium phosphate and calcium sulfate, the degradation speed of it matchs with normol bone, it is as hard as normol bone and three times of cancellous bone,it can be completely degradation in vivo, also it’s a kind of good material for bone transplantation. In this study, we will mix BMSCs,geneX,TGF-β2and culture in vitro.Then discuss the ability of histocompatibility and the influence of proliferation and differentiation of geneX,in order to get some powerful evidence for further study and clinical research in the early treatment stage of femoral head necrosis.Methods:1.BMSCs’s isolationand culture:Healthy New Zealand rabbits’s bilateral femoral shaft were removed in strict compliance with the principles of aseptic.Using adherent method can remove the suspended cells which can’t adherent in order to separate and purificate cells,according to BMSCs can beadherented while blood cells suspended instead of it in the culture medium, we can achieve the purpose of purfying BMSCs through regularex change of medium. Placed in an incubator,For3-4days observed and changed forfluid, passaged the cells after the full growth of the cells and this is denoted as P1generations; we can obtain a more pure adherent cells after subculture. Using immunohistochemistry adherent cells’s surface markers CD44can confirm the resulting cells of BMSCs when spread P3generation,BMSCs is inrelatively high purity, and have form stable, soselect this generation of cells used for subsequent experiments.2.geneX scaffold production:Producing geneX scaffold with strict asepticin a sterile workbench, removing a geneX artificial bone powder syringe as A,another a 2ml syringe of sterile saline as B,Connecting to two syringes with aconnector follow the instructions, injecting saline which in the a syringe B into via syringe A through the connector, so that saline and geneX artificial bone powder can mix into a pulpy, then A tube of artificial mashed geneX injected back into the B tube, mixing artificial mashed geneX so quickly back and forth in the two syringe(about1minute to complete), and finally hit the mashed geneX bone material to A tube, then B tube and connectors were carefully removed from the A tube, remove the discharge connector and connected to the injection syringe A pipefitting, then played38model which is about3mm×2.5mm size and shape similar to the columnar geneX on sterile clean and dry hole shape model, then remove ascaffold material geneX to observe in electron microscopy,the remaining37geneX model immersed in DMEM medium to prepare to use the next compoundand in vitro.3. Composite geneX with BMSCs:Taking osteogenic cultured BMSCs, digesting with0.25%and washing with DMEM containing10%FBS culturetwice, and then suspendedin serum-free culture medium to a final concentration of2×104/ml,inoculated to a good handle37geneX scaffold, making geneX scaffold just been infiltrating in BMSCs cell suspension.Culture the geneX with BMSCs which is composited in the same culture flasks.medium was changed every other day.Turning over the surface of the bone for cloning under aseptic conditions after5days, the cells can be more evenly distributed in the human geneX bone scaffold surface to prepare for the next stepin vitroand remove apiece of geneX scaffold structure observed in the electron microscope.4. geneX and BMSCs in vitro culture for group and detection:Concluding that under lng/ml of TGF-β2is to promote the optimal concentration of BMSCs into bone in the former experiment, adding45ml BMSCs cell culture medium to every bottle The P3cells were divided into three groups, group A containing45ml spread among three generations of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation medium-groups; Group B between adding45ml solution containing bone marrow mesenchymal stem cells (BMSCs)+geneX scaffold osteogenic differentiation medium-groups; group C containing between adding45ml of bone marrow mesenchymal stem cells (BMSCs)+geneX scaffold material+5ml concentration of lOng/ml of TGF-β2in osteogenic differentiation medium-groups (the TGF-β2concentration in the solution is lng/ml). A total of54bottles of inoculation, each inoculated with18bottles,and taking54samples. Control group and experimental group were cultured under the same environment. Taken7days,14days and21days at three time points of BMSCs culture media for different groups of cell morphology, and alkaline phosphatase (ALP) activity assay, MTT detection methyl thymol blue cells was detected in calcium content and calcium Sin red pigment accumulation of staining. Result1. The BMSCs observation of cell morphology:Bone marrow mesenchymalstem cells (BMSCs) cultured in strict aseptic operation after48hours the cells can adherent growth, similar to fibroblast, about96hours later, cells form into long shuttle shape and a lot of cells adherent, began to form a colony, change the culture solution of cells many times, when at the third passage, the purified cells were detected CD44(+) by immunohistochemistry and cell adherent rate in different time points, the results show that our obtain bone marrow mesenchymal stem cells by isolation and identification, and the purity reached95%, that can do backup for subsequent experiment2.BMSCs osteogenic induction culture:With the purity of95%bone marrow mesenchymal stem cells were cultured in osteogenic induction differentiation medium, with the extension of time, cells showed aggregation growth, complex layer growth, no contact inhibition, obvious formed round or ovoid nodules of calcification after3weeks, cells mostly radial distribution at nodules surrounding.3.Observe the geneX scaffolds and composited geneX and BMSCs by the scanning electron microscope:compare both visible BMSCs of osteogenic induction differentiation and geneX scaffolds composited after5days, cells stent adherent gro-wth in the mesh of scaffolds surface, fibroblast growth, shows good histocompatibi- lity.4. Cell growth kinetics MTT detection, the experimental results show that:Bone marrow mesenchymal stem cells were growed in the osteogenic differentiation induction medium, but the higer value of composite BMSCs with geneX artificial bone scaffold group than the only BMSCs group in optical absorption, and bone marrow mesenchymalstem cells (BMSCs)+geneX artificial bone+lng/ml TGF-β2group have highest light absorption degree. B group was higher than that in A group, and C group in the OD value were highest in the three groups at different time points.5.Alkaline phosphatase (ALP) were detected bone marrow mesenchymal stem cells osteogenic differentiation ability in ever group, the experimental results show that:alkaline phosphatase values of A group (only containing BMSCs group) has been in a lower expression level than the other group, and the ALP of other group expression were significantly elevated compared to A group, further comparison,the C group’s alkaline phosphatase activity was the highest.6.Methyl thymol blue (MTB) method for the determination of free calcium, application methyl thymol blue (MTB) method for the determination of free calcium, calcium concentration of B, C group are higer than the control group A, and calcium ion concentration of C group were the highest.7.Alizarin red staining can be observed calcium accumulation under the microscope, B group and C group more than group A in the calcium accumulationare, and calcium accumulation of group C was the highest, that of bone marrow mesenchymal stem cells(BMSCs)+geneX artificial bone+lng/ml TGF-β2group was in most calcium nodules.Conclusion1. In experiments obtain bone marrow mesenchymalstem cells by cell isolation and culture, verify that the BMSCs has very strong osteogenic differentiation ability,it is the ideal seed cells for bone tissue engineering;2. GeneX artificial bone is a good carrier scaffold materials, and composite with BMSCs has good biocompatibility, no toxicity, no inhibitory effect of BMSCs on the osteogenic activity.3. Composited BMSCs and geneX artificial bone can promote the osteogenic activity of BMSCs, promote bone formation rate, proved the promoting effect of TGF-β2to osteogenic activity once again. |
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