| ObjectiveTo investigate the capacity of xenogeneic cancellous bone scaffold combined withNew Zealand white rabbits’s bone marrow stem cells(BMSCs) that induced intoosteoblast in vitro to construct tissue engineering bone in repair of the defect of rabbitulna. To seek the ideal source of seed cells and appropriate scaffold applied in bonetissue engineering, so as to explore a new method to treat bone defects.MethodsCalvarias were dissected from newborn New Zealand white rabbits in24hours，andosteoblasts were isolated with modified tryptase digestion method; BMSCs weregained from long bone of two-week old New Zealand white rabbits with densitygradient centrifugation. The morphology of cells was observed with inverted phasecontrast microscope and the cell activity and cell growth curve of primary cell weredetermined by tetrazolium salt colorimetry; HE cell staining, collagen I and collagenIII immunohistochemical staining and alizarin red staining were used to identify OBand BMSCs.The well third generation OB and BMSCs were co-cultured with transwell chamberand divided randomly into A, B and C group respectively. In the three groups BMSCswere inoculated in lower chamber, group A OB was inoculated in upper chamber,group B was used with osteoblast induction culture medium,and group C was used with BMSCs culture medium. The osteogenic induced cells were identified bymorphological observation, alizarin red staining, alkaline phosphatase and osteocalcindetect in cell culture supernatants,and RT-PCR method detect the expression of OCNand collagen I mRNA.Xenogeneic cancellous bone oriented from quarantined pig femoral condyle wereprocessed into bio-based heterogeneous cancellous bone.Cancellous bone weresoaked with BMSCs culture medium for6h before use, after drying combined withosteogenic induced BMSCs and co-cultured for one week in vitro. The osteogenicinduced cells adhesion and proliferation condition on the xenogeneic bone scaffoldswere detected by MTT method and observed by inverted phase microscope.Thirty-six6months old New Zealand white rabbits were selected with completelyrandom sampling method, thirty rabbits were divided into two groups each is15asexperimental and control group, the rest6rabbits as blank control group, then choseone side of rabbit upper limbs randomly to make15mm segmental bone defect modelin rabbits ulna. According to different implant prosthesis divided into three groups,experimental group: xenogeneic cancellous bone combined with osteogenic inducedBMSCs; control group: xenogeneic cancellous bone only; blank control group: withnothing implant materials.5rabbits from each group were killed at the4th,8th,12thweek after operation, and samples were detected by X-ray radiologicalexamination,general observation and bone defect area histological analyses, and bonedefect area anatomical measurement at12th week after operation, and they were usedto evaluate the healing condition among the three groups.Results1. Extraction and identification of cellsDensity gradient centrifugation was an easy method to obtain high-purity BMSCs,andthe cells presented with long-spindle shape; Modified tryptase digestion method costless time and presented higher production of osteoblasts, and the cells presented withelongated or polygonal shape.The two kinds of primary cell were identified asBMSCs and osteoblasts.2. Osteogenic induction of BMSCsThe AKP and OCN content in the supernatant at the same time testing point detection results showed no significance difference between transwell co-culture method andosteogenic induction medium culture method (P>0.05). The osteogenic induced cellsof the two groups Alizarin red staining were both positive,and the expression of OCNand collagen I mRNA were both positive when detected by using RT-PCR method．3. The preparation of xenogeneic cancellous bone and co-culture with osteogenicinduced BMSCsXenogeneic cancellous bone was made through antigen-free treatment,and presentedwith white or yellow color and porous structure. The osteogenic induced BMSCsgrew well on xenogeneic cancellous bone when observed by microscope,and cellcount increased with the time according to the MTT results.4. The experiment of bone defect repairCallus quantity in defect area and Scores of X-ray of the experimental group werehigher than that of the control and blank group at4,8,12weeks after implantation (P<0.05).Histological observation shows that woven bone mass of the experimentalgroup was more than that of control and blank group (P<0.05). Coronary diameter,sagittal diameter and thickness of the callus of the experimental group were higherthan that of the control and blank group at12th week after operation(P<0.05).Conclusion1. Density gradient centrifugation method and modified tryptase digestion method arereliable and efficient way to obtain BMSCs and OB original generation cells in vitro.2. BMSCs can be induced into osteoblasts by using transellcell chamber, which cannot be used as large-scale culture.3. Xenogeneic cancellous bone have good tissue compatibility with osteogenicinduced BMSCs, which was proved to be a fine scaffold for bone tissue engineering.4. Osteogenic induced BMSCs combined with xenogeneic cancellous bone have goodrestoration function and is examined to be a new method to repair bone defect... |
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