| Objective: Based on previous studies that has proved PBMSCs has therapeutic effectson rabbit ONFH which induced by frozen liquid nitrogen, this study was to prove whetherPBMSCs could survive in vivo and the expression profile of cytokines in the rabbitfemoral damaged area after transplantation were also detected. Thereby to provide somenew and valuable evidence to explain the possible mechanisms of PBMSCs for ONFHtherapy.Method:1)Isolation, culture and immunophenotype identification of rabbit PBMSCs: thestudy used2to3months aged Japanese white rabbit. Firstly, growth factor G-CSF wasused to mobilize the rabbit for6days with30μg/(kg·d) injection subcutaneouly,40mlperipheral blood of the rabbit was aspirated on the7th day, and the peripheral bloodmononuclear cells (PBMNCs) were separated by density gradient centrifugation. Then,using the RBC lysis buffer to eliminate RBC. The modified α-MEM that contains15%FBS was used for PBMSCs primary culture in a humidified atmosphere of95%air and5%CO2at37°C. The culture medium was changed when needed and the other cells wereeliminated. When the adherent cells growth to reach80%~90%confluence, subculture wasperformed with a cell density of2~5×105cells/ml. Finally, the phenotypes of passage2PBMSCs was identified by immuocytochemistry stain CD90, CD44, CD105, CD34, andCD45respectively.2)Establishment the ONFH model of the rabbit: sodium pentobarbitalwere intravenously injected into ear vein of3to4months Japanese white rabbits at thedose of30mg/kg, gluteus muscles were bluntly separated aseptically to expose the femoralhead. Afterwards, frozen and rewarmed the weight loading area of bilateral femoral headcartilage surface several times by sterilized medical cotton stickers dipped with liquidnitrogen.3)Groups dividing of experiment and PBMSCs transplantation: the rabbits weredivided into normal group, model group, core decompression group and PBMSCs transplantation group., In core decompression group, drilled with broach deep into5mmuntil arriving at articular cartilage with30°of right femoral neck, reset the head of femurafter core decompression. In transplantation group, resuspended cells with300μl normalsaline containing cells in the total number of3-4×106of passage2-3PBMSCs was injectedin situ after core decompression. Sutured the incisions of all groups after operation.4)Tracking the survival of the PBMSCs labelled with PKH26in vivo and pathologicalobservation of the damaged area of the femoral head: randomly selected the femoral headof each group on the2nd week,4th week,6th week, and8th week post-intervention, splitthe coronal section of the femoral head. The selected femoral head sample was used tofixed, decalcificated, and splited into frozen sections (15μm) and paraffin sections (10μm)respectively. Then, DAPI double labelling the frozen sections, and observe the survival oftransplanted PBMSCs under the fluorescence microscope. The paraffin sections weremasson stained in order to observe the histopathologic changes under the opticalmicroscope.5)The changes of the cytokines expression of the transplanted PBMSCs withinthe internal femoral head: femoral head sample was stored in liquid nitrogen jar and usedfor real time polymerase chain reaction(RT-PCR) to analyze the levels of BMP-2, BMP-4,PDGF-β, TGF-β1, VEGF-A and CCL2mRNA expressions.Results:1)The morphology and phenotypic properties of rabbits PBMSCs: themorphology of primary cultured PBMSCs was emerged colonies in different sizes at the7thor8thday. The morphology of cells consist mainly fusiform shape, polygonal shape andirregular shape. On the approximately10thday culture, cells grew to approximately80%confluence; the morphology of subcultured PBMSCs was uniform for swirling growth.Immuocytochemistry stain results demonstrate PBMSCs positive expression ofmesenchymal markers CD90, CD105and CD44, but negative expression of hematopoieticcells markers CD34and CD45.2)The analysis of the survival of PBMSCs in damaged area:the results under fluorescent microscope demonstrate the positive transplanted cells labeledPKH26existed in the femoral transplanted area after2weeks,4weeks,6weeks and8weeks transplantation. With the time pass by, the number of transplanted PBMSCsdecreases.3)The pathological analysis of the femoral masson stained tissue: under the microscope, the normal group’s bone trabeculae is complete under the area of cartilage.The arrangement is regular and intensive, and most mature bone tissue stained red.2weeksafter transplanted, the model group’s bone trabecular fracture and the arrangement isdisordered and irregular, and most bone tissue stained green. The number of empty nonelacunae increased within the bone trabeculae. The bone tissue stained green in coredecompression group as well as PBMSCs transplantation group. After4weeks, the bonetrabeculae in model group is thinner than normal one. Bone tissue is stained asymmetrical，but the new born tissue starts to increase. However, the bone trabeculae showshomogeneous stained green with some parts of stained red for both core decompressiongroup and PBMSCs transplantation group.2weeks afterwards, most part of the bonetrabeculae under cartilage area are stained green new bone tissue. The strip of greenstaining bone trabeculae can be seen under core decompression group as well as PBMSCstransplantation group. At the8thweek, the red stained mature bone tissue of model groupincreases, the bone trabeculae is sparse and the sclerotin of cartilage is relatively thin.However, the stained red area in core decompression group expands, the bone trabeculae issparse and the sclerotin of cartilage is relatively thin as well. For the PBMSCstransplantation group, the bone trabeculae shows mixed red and green, and more intensivecomparing with other two groups. The red stained area are mixed with the green stainedarea.5)The results of cytokines mRNA expression: The BMP-4mRNA expressions werelow in the model group(P<0.01). The expression levels of BMP-4mRNA in decompressedpresented higher than the model group(P<0.01) at week4,6and8. At the time point of2weeks, however, the expression level of BMP-4mRNA decompressed group higher thanthat of decompressed group (P<0.05); The BMP-2mRNA expressions were low in themodel group(P<0.01). The expression levels of BMP-2mRNA in decompressed grouphigher than the model group(P<0.01); The PDGF-β mRNA expressions were low in themodel group(P<0.01). The expression levels of PDGF-β mRNA in decompressedpresented higher than the model group(P<0.01) at week6and8. At the time point of4and8weeks, however, the expression level of PDGF-β mRNA decompressed group higherthan that of decompressed group (P<0.05); The TGF-β1mRNA expressions were low in the model group(P<0.05). The expression levels of TGF-β1mRNA in decompressedpresented higher than the model group(P<0.01) at week2,4and8. At the time point of4weeks, however, the expression level of TGF-β1mRNA decompressed group higher thanthat of decompressed group (P<0.05); The VEGF-A mRNA expressions were low in themodel group(P<0.01). The expression levels of VEGF-A mRNA in decompressedpresented higher than the model group(P<0.05) at week2,6and8; The expression levelsof CCL2mRNA in model group were more than normal group(P<0.05), whereas, theexpression of the CCL2mRNA in decompressed group, was obviously lower than modelgroup at week2,4and8(P<0.05). Furthermore, the expression of the CCL2mRNA intransplanted group was lower than decompressed group at every time point(P<0.05orP<0.01).Conclusion: PBMSCs could survive at least8weeks in situ of ONFH. The recoveryeffect of ONFH after PBMSCs transplantation might relate to the changes on theexpression of cytokines such as BMP-2, BMP-4, PDGF-β, TGF-β1and VEGF-A that takepart in both the recovery of damaged bone tissue and improvement of the blood supply. |
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