Artificial Extracellular Scaffold:the Effect Of Three-dimensional Structure Of50μm Titanium-web(TW) On The Differentiation Of Rabbit Bone Marrow Mesenchymal Cells Into Osteoblasts

Bone defect is still the currently serious problem cause function lossand lower living quality of patients. Autologous bone graft is still the mostcommon method in clinic to repair bone defect by far, but it is associatedwith undeniable drawbacks. Seeking for the better method to repair bonedefect become the research focus of today, which requires the efforts ofmultidisciplinary. The rise of tissue engineering provides a broad way ofrepairing bone defect. In the1980s, the mesenchymal stem cells with thepotential of multiple differentiation were found, which makes it to be theideal seed cells in bone tissue engineering. And the scaffold materials alsogradually shift from two-dimensional to three-dimensional structure.Titanium web with the characteristic of three-dimensional structure wasproposed in this study. Rabbit bone marrow mesenchymal stemcells(rBMSCs) were cultured in vitro to observe the effect of titanium webon the differentiation of rBMSCs into osteoblasts. In order to understand the characteristics of this material for bone tissue engineering scaffoldmaterials.Materials and Methods1. The titanium webs (TW) were designed by Japanese scientistYoshinori Kuboki and produced by Japanese company HI-LEX. The TWconsisted of fibers with diameter of50μm which could provide a suitableenvironment for bone ingrowth in vivo. The dimension of the TW is5mm,the thickness is1.5mm,and the porosity is87%.Common level ofNew Zealand white rabbit,4~6weeks were selected. Injected20%urethane according to5ml/kg dose in New Zealand white rabbit foranesthesia, and took out the femur in aseptic conditions then moved it tobechtop. Cut along the middle femur, rushed the contents of the medullarycavity out over and over again. The method combined with density gradientcentrifugation and whole bone marrow adherent culture was used to isolatethe rBMSCs.2. Identification of rBMSCs. Growth characteristics were observed byinverted microscope, the third generation of cells was selected, achieving80%fusion, to carry out osteoblastic induction and lipoblastic induction.Two weeks later, we observed whether the directional differentiation ofcells occurred by ALP staining and oil red O staining to determine whetherthe cells cultured in vitro were rBMSCs.3. rBMSCs were cultured together, using the inverted microscope to observe if the cells could grow on the TW successfully.4. Due to opaqueness of TW, growth characteristics of cells on TWcould not be observed accurately by inverted microscope. So the scanningelectron microscopy (sem) was used to observe.5. The TW was taken out after co-culture of TW and rBMSCs, and theanalysis of the effect of TW on rBMSCs was carried out by ALP staining.Results1. In the early stage of the culture, a few mononuclear cells were isibleby inverted phase contrast microscope. And the morphology of cells waspolygon. After6-10d, cells proliferated quickly and cell fused, morphologybecame long fusiform as fibroblasts and homogeneous as fish.2. Osteoblastic induction and Lipoblastic induction of the primary cellwas carried out: the alizarin red staining was carried out after2weeks ofosteoblastic induction, mineralization nodules were found, and there werelots of palm red granules found in the endochylema by ALP staining. After2weeks of Lipoblastic induction, the cells became bigger and emptybubbles were emerged surrounded the nucleus. There were lots of red lipiddroplets after oil red staining.3. The relationship of cells and TW was observed by inverted phasecontrast microscope, because of the opaqueness of TW, we couldoccasionally find a few cells on the edge of TW. But through scanningelectron microscopy (sem), it was easy to find the cells on TW.1week after co-culture of rBMSCs and TW, there were cells on the surface of thematerial, but the number is small. With the extension of time, the cell grew,proliferated and differentiated.2weeks later, a large number of cellsadhered on TW, it was apparently more than1week. The morphology ofthe cells was long spindle, reticularly covering the surface of the material.The secreting matrixes of the cells were deposited around the cells and thesurface of the scaffold.4.2weeks after co-culture of rBMSCs and TW, ALP staining wascarried out, there were lots of palm red granules in the endochylema, andthe granules were gathered as linear. But in the control group, it was weakpositive, there were only few granules, and the granules were scattered.Conclusion1. Using the method combined with density gradient centrifugationand whole bone marrow adherent culture can isolate and culture rBMSCssuccessfully in vitro.2. The artificial extracellular scaffold with three-dimensional titaniumweb of50μm filaments is non-toxic to rabbit bone marrow mesenchymalstem cells (rBMSCs),and the cells can grow on the scaffold very well. Thescaffold of titanium web could also induce the cells to be osteoblasts, this will provide some experimental basis and support to make it to be an idealrepair material in bone tissue engineering.