Combined Application Of Green Fluorescent Protein Labeling,SEM And X-Ray Photoelectron Spectroscopy To Observe Mineralization Of Rabbit BMSCs In Vitro

Objective: To observe the morphological changes of rabbit bone-marrow mesenchymal stem cells(BMSCs) after osteogenic induction and calcified plaque formation of osteogenesis of rabbit BMSCs labeled by green fluorescent protein(GFP)in vitro and surface microstructure and quantitatively analyze the elements of calcified plaque by scanning electron microscope and energy dispersive spectrometer(SEM/EDS), and to observe surface microstructure and biological mineralization of tissue engineering bone by GFP labeling and SEM/EDS. Methods: The rabbit BMSCs were traced by adenovirus with GFP(Ad-GFP) gene, and then cells were induced differentiation to osteogenesis.Inverted fluorescence microscope and alizarin red staining were applied for observing morphological changes of cells after transfection and calcified plaque formation and SEM/EDS were employed to study the surface microstructure and element composition of calcified plaque. The BMSCs were marked by Ad-GFP.Real-time growth of the cells were observed by an inverted fluorescence microscope after the osteoinductive culture on to Decalcified bone matrix(DBM), and surface microstructure and biological mineralization of tissueengineering bone were observed by SEM and X-Ray photoelectron spectroscopy. Results: Fluorescence microscope showed rabbit BMSCs after Ad-GFP transfection gave green light and the cell morphology differentiated to osteogenic accompanied with opaque mineralized calcium nodule. Alizarin red staining exhibited red mineralized nodules. From SEM, punctate mineralization calcium nodules scattered among cells and cells grew overlapplystrongly secreted matrix. EDS confirmed that the main elements of mineralized calcium nodules were analogous to normal bone tissue and ratio of calcium to phosphorus(Ca/P) was 1.55, close to normal ratio 1.49. The cells on surface of DBM had a good adhesion, overlap growth, as observed by an inverted fluorescence microscope, and a higher level of transient expression of GFP was confirmed after 14 days in vitro culture. SEM image showed that DBM had a porous structure, with pore diameter ranging from 300 to 600 μm, and a porosity rate was around 90%. The tissue engineering bone were showed that cells grew adherently on the surface of DBM, and matrix secretion was strong, and the DBM was covered by rough biological mineralization. X-Ray photoelectron spectroscopy showed that the surface of rough biological mineralization consisted of calcium、phosphorus sediment, and its Ca/P was 1.46. Conclusions:Rabbit BMSCs can be successfully transfected and labeled by Ad-GFP and trend to osteogenic differentiation after osteogenesis induction; BMSCs have a preferable osteogenic capability and the process of biological mineralization in vitro is the same with in vivo;Tissue engineering bone constructed by DBM scaffold materials in vitro has exc ellent biological properties, and combined application of GFP labeling; SEMand X-Ray photoelectron spectroscopy is a feasible method for evaluating DBM scaffold material in tissue engineering bone.